Radioimmunotherapy of lymphoma using anti-CD20

ABSTRACT

Methods for the treatment of lymphoma by adminstration of a B cell-specific antibody are described. The invention encompasses providing to a patient both unlabeled antibodies and antibodies labeled with a radioisotope. A principal advantage of the method is that tumor responses can be obtained in a radiometric dose range that does not require hematopoietic stem cell replacement as an adjunct therapy.

FIELD OF THE INVENTION

The invention relates to therapy of lymphoma using antibodies directedto an antigen present on the surface of the lymphoma cells. The antibodydemonstrates a therapeutic effect when administered per se, however,greatly enhanced therapeutic effect is seen when the antibody is labeledwith a toxic substance, e.g. radioactively labeled. The amount ofradioactivity used to label the antibody is preferably low enough thattoxicity to bone marrow and other tissues is avoided, yet high enough toeffect complete remission of the lymphoma.

DESCRIPTION OF RELATED ART

Although significant advances have been made in the treatment ofnon-Hodgkin's lymphoma over the past two decades, a curative regimen forpatients with low-grade B cell lymphomas has yet to be developed. Inaddition, durable remission in patients treated with various regimensfor refractory intermediate- and high-grade lymphomas have beenrelatively rare (1). Recent attempts utilizing supralethal chemotherapycombined with radiotherapy followed by bone marrow transplantation haveresulted in an approximately 20% long term disease-free survival rate(2). However, most patients treated in this manner die of lymphoma ortreatment related complications. Therefore, new strategies for thetreatment of non-Hodgkin's lymphomas are needed. These strategies shouldhave as their goal the maximization of therapeutic effect coupled withthe minimization of toxicity.

One approach involves the use of monoclonal antibodies which recognizetumor-associated antigens as a means of targeting drugs or radioisotopesto tumor cells. This approach is particularly attractive in the case ofnon-Hodgkin's lymphomas as the tumor cells of these lymphomas display avariety of tumor-restricted antigens on their cell surfaces which wouldbe available for targeting (3).

The rationale for utilizing such an approach is further supported by theobservation that monoclonal antibodies by themselves can exhibitantitumor effects in vivo. Of all the malignancies that have beentreated with monoclonal antibodies to date, the lymphomas have yieldedthe most dramatic results. In particular, significant tumor regressionshave been reported in patients treated with monoclonal anti-idiotypeantibodies (4,5). Most of the tumor responses, however, have beenincomplete and of relatively short duration. The practical problem ofgenerating anti-idiotype antibodies specific for each individualpatient's idiotype and the emergence of idiotypic variants duringanti-idiotype therapy (6) restricts the utility of such an approach.

In light of these findings, it is worth considering whether lessrestricted antigens on lymphoid tumor cells might be appropriate targetsfor therapy. In general, anti-tumor effects of antibodies against suchantigens have only been modest. Patients with chronic lymphocyticleukemia (CLL) and cutaneous T-cell lymphomas, for instance, have beentreated with the T101 antibody which binds a 65 Kd glycoprotein presenton malignant and some normal T-cells (7). Transient reductions incirculating malignant cells in CLL patients and temporary improvementsin skin lesions in cutaneous T-cell lymphoma patients, have beendemonstrated (8-11). Recently, a number of murine monoclonal antibodieshave been developed which recognize antigenic sites on both malignantand normal human B cells (12-19). These pan-B-cell antibodies have beenuseful in classifying lymphomas and in defining the ontogeny and biologyof normal B cells. Therapeutically, these antibodies have principallybeen used in ex vivo purging of autologous bone marrow of malignantcells prior to bone marrow transplantation (20-22). The limitedexperience with these antibodies as therapeutic agents in vivo hasindicated only modest activity (22, 23).

Because of the limited efficacy of unmodified antibodies in general,recent attention has focused on the use of antibodies conjugated tocytotoxic agents. Among the cytotoxic agents which might be considered,radioisotopes are especially attractive, as lymphomas are especiallysensitive to the effects of radiation. Moreover, such radiolabeledantibodies may be of considerable utility in terms of diagnostic imagingof tumor involved sites. Imaging trials have been carried out using ¹¹¹In and ¹³¹ I conjugated to T101 antibody, for example, in patients withCLL and cutaneous T-cell lymphoma (24). Intravenous administration of¹¹¹ In-labeled T101 was shown to be capable of detecting tumors as smallas 0.5 cm in diameter. These studies also demonstrated that isotopelocalization to tumor could be achieved despite the presence of targetantigen on normal as well as malignant cells.

The therapeutic potential of radiolabeled antibodies in lymphoma hasrecently come under investigation. Badger et al., using a murine T-celllymphoma model, have demonstrated that a monoclonal antibody against theThy 1.1 differentiation antigen labeled with ¹³¹ I was superior tounmodified antibody in its therapeutic effect (25). The dose limitingtoxicity in these experiments was that of bone marrow suppression. Rosenet al, have reported their results using ¹³¹ I-labeled T101 antibody inthe imaging and therapy of 6 patients with cutaneous T-cell lymphoma inwhich significant responses of disease lasting 3 weeks to 3 months wereobserved (26). As in the murine model of T-cell lymphoma,myelosuppression was again seen as the dose limiting toxicity in thesepatients.

Since greater than 75% of all non-Hodgkin's lymphomas are of B celllineage, we and others have begun to investigate the use of pan-B-cellmonoclonal antibodies labeled with radioisotopes in preclinical andclinical studies. We have been able to demonstrate, for instance, usinga nude mouse model of xenografted human B cell lymphomas, thatradiolabeled pan-B-cell antibodies can be specifically targeted to Bcell tumors in vivo (27) and that these radiolabeled antibodies can havetherapeutic effects. DeNardo et al. have reported their experience with¹³¹ I-labeled Lym-1 antibody (28). Lym-1 is an IgG2a antibody whichrecognizes a cell surface antigen of 31-35 Kd, which appears to be anHLA-Dr antigen, and reacts with normal and malignant B cells (29).

Recently, we performed a study using the pan-B-cell antibody MB-1labeled with radioiodine as a radioimmunodiagnostic and therapeuticagent. MB-1 is an IgG1 anti-CD37 monoclonal antibody, which binds to Bcells bearing the 40 Kd cell surface protein CD37. MB-1 binds to almostno pre-B cells (30). This antibody has been found to also react withgranulocytes, platelets, and T cells, but the magnitude of this bindingis less than the binding to B lymphocytes. No binding has been observedwith tissues from stomach, thyroid, kidney, skin, peripheral nerve,heart, and cervix. In a study, twelve patients with refractory B celllymphoma were evaluated for the biodistribution of ¹³¹ I-labeled MB-1,its imaging potential, toxicity, and therapeutic effect. Successfulimaging of tumors has been achieved in all but one of our patients, butnot all known tumor sites were visualized in all patients. Significantclinical responses have been documented, although only one completeresponse and one partial response were achieved at the dose levelsemployed. Also, severe myelosuppression precluded further doseescalation. Press et al. have reported their experience with ¹³¹ I-MB-1using higher radioactivity and protein doses than those we employed inour trial (31). Four patients have been treated with single doses ofbetween 232 and 608 mCi of iodinated antibody combined with large dosesof antibody (2.5-10 mg/kg total antibody) with provision for autologousbone marrow rescue. Each of these four patients obtained a completetumor remission. Severe myelosuppression occurred in all patients,however, with two patients requiring reinfusion of previously storedautologous bone marrow. No other significant acute toxicity was seen.Two patients relapsed with lymphoma 4 and 6 months after achievingcomplete remission and the remaining two patients remain in continuousremission at 8 and 11 months.

It is presently unclear why a substantial number of patients will notreceive radiation doses to all tumor sites which are significantly abovethose doses given to normal tissues using Lym-1 or MB-1. Whether this isdue to the nature of the antibody being utilized, the stability of theradiolabel in vivo, the method of administration, the overall tumorburden within the host, or other factors related to the tumor or itsvasculature remains to be determined. One factor which should beseriously considered is the cross-reactivities encountered with thesetwo antibodies with either normal non-lymphoid tissues or otherhematopoietic cells. The biodistribution patterns of antibodies withmore restricted specificity for B cells might be more favorable in thatnonspecific absorption in vivo might be reduced.

One antibody that is somewhat more specific for B cells is the antibodyLL2. A clinical study of radioimmunotherapy of lymphoma using labeledLL2 has been reported, but the results were somewhat disappointing, inthat of only one of the five patients assessed exhibited a completeresponse, two patients exhibited a partial response, two exhibited aminor or mixed response, and severe myelosuppression was encountered(63).

CD20 is an antigen that is a 35 kilodalton, non-glycosylatedphosphoprotein found on the surface of greater than 90% of B cells fromperipheral blood or lymphoid organs. The antigen is expressed on thesurface of virtually all resting B cells maintained in culture, but islost by approximately one-third of the population upon activation of thecells by protein A or exposure to Epstein-Barr Virus. This result hasbeen interpreted to mean that CD20 is lost during terminaldifferentiation of B cells (74). The antigen bound by LL2 shows asimilar distribution to CD20, but is distinguishable by virtue of alower antigen density on the surface of B cells for LL2 than for CD20(77).

The 1F5 antibody against CD20 has been previously used in studies ofradioimmunotherapy of lymphoma (31). Again, the results of this studywere disappointing, in that only partial regression of the lymphoma ofthe treated patient was observed.

Another anti-CD20 antibody is the antibody anti-B1 (hereafter referredto as B1). B1 is an IgG2a that immunoprecipitates a 35 Kd cell surfacephosphoprotein (CD20) expressed by normal B cells in various stages ofdifferentiation, follicular and diffuse B cell lymphomas, and variouslymphoid leukemias (32). No reactivity of this antibody has beendemonstrated with granulocytes, platelets, thymus tissue, or T cells.

A significant amount of information is now available regarding the CD20antigen which B1 recognizes. It is apparently expressed early in pre-Bcell development just before the expression of cytoplasmic μ heavychains and persists until plasma cell differentiation. The binding of B1to the extracellular portion of the CD20 antigen generates atransmembrane signal which can inhibit the cell's entry into the S/G2+Mstages after mitogen stimulation and also blocks differentiation intoantibody-secreting cells (33-36). Antagonistic effects on B cellactivation have also been observed with B1 binding and these differencesmay be due to differences in the state of activation of these cellsbefore signal generation (37,38). Of interest are data using thisantibody in nude mice bearing B cell lymphoma xenografts (39) andimaging performed in Rhesus monkeys using ¹³¹ I-B1 in which the B cellrich spleen could be readily visualized by gamma camera scanning withoutneed for image intensification or background subtraction techniques(36). The predominant use of B1, however, has been in the ex vivopurging of bone marrow prior to autologous bone marrow transplantationin patients with refractory leukemia and lymphoma (40). These studieshave shown that marrow reconstitution is unaffected by the B1 antibody.Thus, B1 is an attractive antibody for use radioimmunodiagnoisticallyand radioimmunotherapeutically.

CD19 is another antigen that is expressed on the surface of cells of theB lineage. Like CD20, CD19 is found on cells throughout differentiationof the lineage from the stem cell stage up to a point just prior toterminal differentiation into plasma cells (74). Unlike CD20, however,antibody binding to CD19 causes internalization of the CD19 antigen.CD19 antigen is identified by the HD237-CD19 antibody (also B4 or theantibody of the B4-89B line, "B4" hereinafter)(92), among others. TheCD19 antigen is present on %4-8 of peripheral blood mononuclear cellsand on greater than 90 percent of B cells isolated from peripheralblood, spleen, lymph node or tonsil. CD19 is not detected on peripheralblood T cells, monocytes or granulocytes. Virtually all non-T cell acutelymphoblastic leukemias (ALL), B cell CLL and B cell lymphomas expressCD19 detectable by the antibody B4 (16, 94).

Additional antibodies which recognize differentiation stage-specificantigens expressed by cells of the B cell lineage have been identified.Among these are the B2 antibody, directed against the CD21 antigen, B3antibody directed against the CD22 antigen and the J5 antigen, directedagainst the CD10 antigen (also called CALLA) (see FIG. 4). Thereactivity of B4 with various tumor types is described above. B2antibody reacts with resting B cells of all lymphoid types and is lostupon activation of the resting B cell. It can be used to identifyheterogeneity in B cell CLL and lymphoma. B3 antibody marks all HairyCell leukemias. The CD22 antigen identified by B3 is found in thecytoplasm of virtually all B cell leukemias and lymphomas. The CALLAantigen identified by J5 is found on 80% of non-T cell ALLs and asignificant portion of B and T cell lymphomas and some T cell leukemias.Of significance to the present invention, CALLA and CD19 are notexpressed by greater than %95 of human bone marrow samples examined(83).

ABBREVIATIONS

The following abbreviations are used in this text:

cGy, centigrays; 1 cGy is approximately 1 rad; C R, CR, completeremission CT, computed Tomography;

DTPA, diethylenetriaminepentaacetic acid;

EDTA, ethylenediaminetetraacetic acid;

MX-DTPA, metal chelate-diethylentraminepentaacetic acid;

mCi, millicurie, 1 mCi=2.2×10⁹ decays per minute;

PR, partial remission;

PD, progressive disease;

RIC, radioimmunoconjugate;

RIS, radioimmunoscintigraphy;

RIT, radioimmunotherapy;

SUMMARY OF THE INVENTION

The present invention provides compositions and articles of manufacturewhich comprise the antibody B1, which binds specifically to the CD20antigen of B cells, and also provides methods for immunotherapy oflymphoma which employ the B1 antibody. In particular, the articles ofmanufacture comprise the B1 antibody and printed matter which indicatesthat the antibody is to be employed in diagnostic imaging and/orimmunotherapeutic methods. Compositions of the present inventioncomprise radioactively labelled B1 antibody and pharmaceuticallyacceptable carriers, diluents and the like. The methods employing B1antibody encompass several embodiments.

One method for using the B1 antibody comprises administeringradiolabeled B1 in a single dose designed to deliver a high amount ofradioactivity. In such a method, it is contemplated that a radiometricdose of greater than 200 cGy is delivered to the whole body of thepatient. In this "high-dose" method, bone marrow transplantation, orsome other means of reconstituting hematopoietic function in thepatient, is required.

In a second method using B1 antibody, a therapeutic dose of radiolabeledB1 antibody is administered, however, the radiometric dose received bythe patient is limited to a level that toxicity to bone marrow is notsignificant and reconstitution of hematopoietic function, by bone marrowtransplantation or other means, is not required. A range of doseeffective in this method is one which delivers between 25 and 200 cGy,preferably 25 to 150 cGy to the whole body of the patient.

A third method using B1 antibody comprises administering to a patient alarge amount of an unlabelled antibody, which can be B1 but can also beother antibodies, prior to administration of a therapeutic dose oflabelled B1 antibody. This therapeutic dose can be made to deliver aradiometric dose of 5 to 500 cGy, preferably, 25 to 150 cGy, to thewhole body of the patient.

A fourth method of using B1 antibody comprises administering atrace-labelled amount of B1 antibody, followed by imaging of thedistribution of the B1 antibody in the patient. After imaging, atherapeutic regime of radiolabeled B1 is administered, designed todeliver a radiometric dose of 25 to 500 cGy, preferably 25 to 150 cGy,to the whole body of the patient.

The doses described above are limits for single administrations. Suchadministrations may be repeated, thus the patient might receive a muchhigher total accumulated dose over the course of imaging and therapy.

It is considered, due to the similarity of the expression of CD20 andCD19 antigen in the B cell lineage, that the methods of the presentinvention can be applied using an anti-CD19 antibody, preferablyHD237-CD19 or B4, in the same manner as is described for an anti-CD20antibody.

Also, the invention is not limited to the CD19 and CD20 antibodies.Rather, the invention encompasses the use of antibodies which areidentify antigens associated with cells of the B cell lineage to treatcancers which are clonal from such cells. Examples of such antibodiesare B2, B3, B4 (HD-237), and J5, in addition to B1. Examples of suchcancers are ALL, CLL, Hairy Cell leukemia, and chronic myeloblasticleukemias in a blast crisis stage, in addition to lymphomas.

Furthermore, it should be noted that the therapeutic method of thepresent invention is amenable to repeated administration for treatmentof chronic disease or relapse after a period of remission. Also, theimaging applications described herein can be applied as diagnosticmethods in their own right. That is, for example, the presence andlocation of CD20 positive cells in a patient can be-determinedindependently of any therapeutic intent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows gamma-camera images of patients with B cell lymphomas afterinjection of ¹³¹ I-labeled anti-B1 antibody. FIG. 1A is an anterior viewof Patient 9 obtained 120 hours after trace-labeled antibody injection.Multiple tumors (arrows) can be seen, 2 to 6 cm in diameter involvingthe neck, the right axilla, and the itiac, inguinal and femoral regions.FIG. 1B is a posterior view of Patient 2 obtained 235 hours aftertrace-labeled antibody injection. There is distinct focal uptake(arrows) within the spleen consistent with intrasplenic tumor targeting.ACT scan of this patient also demonstrated low-attenuation lesions inthe spleen consistent with involvement by lymphoma.

FIG. 2 shows tumor responses to ¹³¹ I-labeled anti-B1 antibody. FIG. 2Ashows abdominal CT images of Patient 4, showing a large chemotherapyresistant retroperitoneal mass before study entry. FIG. 2B showsregression of this mass after one round of radioimmunotherapy. FIG. 2Cshows thoracic CT images of Patient 2 before study entry. FIG. 2D showsCT imaging of the same section six weeks after oneradioimmunotherapeutic dose of 45 mCi, showing the regression ofchemotherapy-resistant mediastinal and peritracheal lymphadenopathy.

FIG. 3A and 3B show a synthetic pathway for(p-isothiocyanatobenzyl)methyl diethylenetriaminepentaacetic acid(mixture of 1-benzyl,3-methyl and 1-benzyl,4-methyl isomers). Thepathway is modified from reference 93.

FIG. 4 shows a representation of the expression of various antigensduring differentiation of the B cell lineage.

DETAILED DESCRIPTION OF THE INVENTION

The principal problem of cancer chemotherapy is achieving goodtherapeutic indices for the compounds administered for the purpose ofkilling the tumor cells. In general, it has been found that tumoricidaldrugs are also toxic to cells of normal tissue, and thus theside-effects of chemotherapy are often almost as devastating to thepatient as the tumor burden itself. Typically, the approach used toachieve some degree of selectivity is to administer compounds which arepreferentially taken up or preferentially toxic to rapidly dividingcells, when compared to their effect on growth arrested cells. Thisapproach is limited by the fact that most, if not all, normal tissuescontain compartments of dividing cells.

The advent of monoclonal antibody technology provided a new means ofproviding selectivity to chemotherapeutic agents. By conjugating thetumoricidal agent to an antibody directed against antigens present ontumor cells, but not present on normal cells, it was expected thatselective killing of tumor cells could be achieved.

Many different conjugates have been created, using antibodies directedto a variety of cell-surface antigens and a variety of tumoricidalsubstances. To date the tumoricidal agents have principally beenradioisotopes and various plant and bacterial toxins. However, whilethere are several reports of individual successes, the results oftherapy using antibody conjugates has generally been disappointing.Remission rates have been low and not generally reproducible.

Lymphomas are tumors of the immune system. They present as both T cell-and as B cell-associated disease. Bone marrow, lymph nodes, spleen andcirculating cells are all typically involved. Typically the initiatingtumor cell is one of the blast cell types later in the lineage, ratherthan an early stem cell. This characteristic has allowed treatmentprotocols wherein bone marrow is removed from the patient and purged oftumor cells, using antibodies directed against antigens present on thetumor cell type, and stored. The patient is then given a toxic dose ofradiation or chemotherapy and the purged bone marrow is then reinfusedin order to repopulate the hematopoietic system of the patient.

The sorting of cells of the hematopoietic lineage, based uponsurface-expressed antigens, is an established art, and severalpopulations of hematopoietic cell types have been defined on that basis.In B cell lymphomas, antibody-targeted therapies have focussed on threeparticular antigens found on the surface of B cells, CD20, CD37 and theHLA-Dr antigens. As described above, therapeutic trials have beenconducted using antibody conjugates recognizing each of these antigens.The method of the present invention employs an antibody directed againstCD20, antibody B1, for the treatment of B cell lymphoma.

Antibody B1 is obtained from the Hall 299-15 cell line, which was firstisolated at the Dana-Farber Cancer Institute. Coulter Clone® B1 can bepurchased from the Coulter Corporation. Details of preparation of theantibody are provided below. The B1 antibody is of mouse origin. As suchit can provoke a "human antimouse antibody" (HAMA) response in humanrecipients, although the frequency of this response is relatively low,especially in patients having B cell malignancies, due to theimmunosuppressive character of the disease. Accordingly, use of anantibody comprising the B1 antigen-binding domain and a human Fc andhinge region is to be considered encompassed by the present invention,as well as alternative methods of "humanization" of the antibody, assuch an antibody would be expected to be less likely to evoke a HAMAresponse, which can be limiting of retreatment of patients with thepresent method. However, HAMA responses occur much less frequently inpersons with B cell malignancies due to the immunosuppressive characterof the diseases. Furthermore, it is expected that it is advantageous toprovide Fab, Fab' or F(ab')₂ fragments containing the antigen-bindingportion of the B1 antibody as the B cell targeting moiety. Such antibodyfragments might provide better diffusion characteristics in vivo, due totheir smaller size, than the whole B1 antibody, in addition to alsobeing less likely to evoke a HAMA response. While F(ab')₂ fragments ofB1 itself are not stable, due to the IgG2a nature of the antibody, anIgG1 constant region variant of B1 could be produced which would formstable F(ab')₂ fragments. The means for engineering of antibodies byrecombinant DNA and chemical modification methods are consideredwell-known in the art.

The B1 antigen (CD20) is present on approximately 9% of unfractionatedperipheral blood mononuclear cells and on greater than95% of normal Bcells isolated from peripheral blood, lymphoid tissues, and bone marrow(12). It is also expressed on tumor cells isolated from 50% of patientswith non-T cell acute lymphoblastic leukemias (ALL), greater than 95% ofpatients with B cell chronic lymphocytic leukemias (CLL), and greaterthan 90% with non-Hodgkin's B cell lymphomas. In contrast it is notreactive with resting or activated T cells, monocytes, granulocytes,erythrocytes, or null cells, and tumors of T cell, myeloid, anderythroid origins. Functional studies demonstrated that the removal ofthe B1 reactive cells by cell sorting or by complement-mediated lysiseliminated all cells from peripheral blood and spleen capable of beinginduced by pokeweed mitogen to differentiate intoimmunoglobulin-secreting cells (80). The B1 antigen appears to beexpressed on all stages of B cell differentiation from the pre-B celland is lost just prior to the development of the plasma cell (80,81).The B1 antigen appears to be distinct from all previously described Bcell determinants including conventional immunoglobulin isotypes,Ia-like antigens, and Fc and C3 receptors. The B1 antigen defines a cellsurface non-glycosylated phosphoprotein of 35,000 daltons (82). The B1antigen does not modulate from the surface of B1-positive cells afterbinding of B1 monoclonal antibody. The B1 antigen is usually found on3-5% of normal human bone marrow cells (80). One to three percent ofthese cells contain intracytoplasmic immunoglobulin (83). Contaminatingperipheral blood B cells in bone marrow account for the additional 1-2%staining and these cells express surface immunoglobulin. More recentstudies have demonstrated that the B1 (CD20) antigen appears in themid-stages of human pre-B cell differentiation (83). The earliest B cellantigens, Ia and CALLA, precede the appearance of CD20 in pre-B cellontogeny. All cytoplasmic μ-positive pre-B cells express the B1 antigen.

B1 anti-CD20 is a murine IgG2a antibody and is capable of mediating invitro lysis in the presence of rabbit complement. Mixing experimentswith normal human bone marrow and tumor cell lines have demonstratedthat anti-B1 can eradicate greater than 99% of tumor cells (84).Moreover, in these experiments there was no toxicity of either B1anti-CD20 or the complement to early differentiated bone marrow stemcells as determined by the CFU-C, CFU-E, BFU-E and the mixed colonyassay (85,86).

B1 anti-CD20 did not decrease the growth of myeloid and erythroidprecursors in a Dexter culture system (86). B1 anti-CD20 antibody, withits specificity for B cells, the lack of modulation of the CD20 antigenafter B1 antibody binds, and its lack of toxicity to the myeloidpluripotent progenitor cells should bind to occult tumor cells of B cellorigin in vivo and deliver a therapeutic radioisotope to the cellsurface of recurrent NHL B cell tumor cells. Normal differentiatedCD20-positive B cells will also bind B1 and be exposed to thetherapeutic radioisotope, but the immature B cell precursor cells arenot CD20-positive. As noted above, the CD19 antigen is expressed in theB cell lineage in a fashion which is very similar to the CD20 antigen.Some of the properties of the CD19 antigen have been describedhereinabove. Additional description of the CD19 antibody can be found inreferences 88-91. From this consideration, one of skill in the art wouldexpect that the methods described in detail hereinbelow using the B1antibody could also be applied using an antibody directed against theCD19 antigen, such as the anti-CD19 antibody, obtained from the cellline HD237-CD19 (Coulter designation MCB 88-8). This cell line wasdeveloped by Dorken et al. at the University of Heidelberg and isdescribed in reference 92. A cell line of the Coulter Corporation,B4-89B, produces a similarly useful antibody.

Additional neoplasms of B cell lineage which can be treated using themethods of the present invention are described above. Also describedabove, and in FIG. 4, are additional antigens (and antibodies againstthem) which are useful targets for imaging and therapy using the methodsof the present invention. Each of the antibodies B2, B3, B4 and J5 canbe purchased from the Coulter Corporation, Hialeah, Fla. Forradiolabeling the antibody, there are several considerations for themethod to be used. First, the radioisotope must be chosen, and then themeans of attaching the radioisotope to the antibody must be selected.With respect to the choice of radioisotope, a general review ofconsiderations is provided by Magerstadt (76). Principally one mustconsider the desired range of emission (affected by parameters includingtissue type of the tumor, whether it is a solid or disseminated tumorand whether or not all tumor cells are expected to be antigen positive),the rate of energy release, the half-life of the isotope as compared tothe infusion time and clearance rate, whether imaging or therapy is theaim of the labeled antibody administration, and the like. For imagingpurposes according to the present invention, it is considered thatlabeling with ⁹⁹ Tc, ¹¹¹ In, ¹²³ I or ¹³¹ I is preferable, with ¹¹¹ Inor ¹³¹ I labeling most preferred. For therapeutic purposes according tothe present invention, it is considered that labeling with a β⁻ emitter,such as ⁹⁰ Y or ¹³¹ I is preferable. In some cases, labelling with an ∝emitter is appropriate. Additional isotopes that merit consideration fortherapeutic or diagnostic uses are ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, ³² Pand radioactive isotopes of Lu.

In considering the means for attaching the radioisotope to the antibody,one must consider first the nature of the isotope. Iodine isotopes canbe attached to the antibody by a number of methods which covalentlyattach the isotope directly to the protein. Chloramine T labeling (87)and iodogen labeling (45) are two commonly used methods of radioiodinelabeling. For isotopes of metals, e.g. ⁹⁰ Y or ¹⁸⁶ Re, the isotope istypically attached by covalently attaching a chelating moiety to theantibody and then allowing the chelator to coordinate the metal. Suchmethods are described, for example, by Gansow et al., U.S. Pat. Nos.4,831,175, 4,454,106 and 4,472,509, each of which is hereby incorporatedin its entirety by reference.

It should be noted that antibodies labeled with iodine isotopes aresubject to dehalogenation upon internalization into the target cell,while antibodies labeled by chelation are subject to radiation-inducedscission of the chelator and to loss of radioisotope by dissociation ofthe coordination complex. In some instances, metal dissociated from thecomplex can be recomplexed, providing more rapid clearance ofnon-specifically localized isotope and therefore less toxicity tonon-target tissues. For example, chelator compounds such as EDTA or DTPAcan be infused into patients to provide a pool of chelator to bindreleased radiometal and facilitate excretion of free radioisotope in theurine. Also, it merits noting that free iodine, resulting fromdehalogenation, and small, iodinated proteins are rapidly cleared fromthe body. This is advantageous in sparing normal tissue, including bonemarrow, from radiotoxic effects.

Methods of administration are also reviewed by Magerstadt (76). Fortreatment of lymphoma, it is considered on the one hand that intravenousinjection is a good method, as the thoroughness of the circulation inrapidly distributing the labeled antibody is advantageous, especiallywith respect to avoiding a high local concentration of the radiolabel atthe injection site. Intravenous administration is subject to limitationby a "vascular barrier", comprising endothelial cells of the vasculatureand the subendothelial matrix. Yet, it is also noted that this barrieris a larger problem for uptake of labeled antibody by solid tumors.Lymphomas have relatively high blood flow rates, contributing toeffective antibody delivery. In the case administration for thetreatment of lymphoma, consideration should also be given tointralymphatic routes of administration, such as subcutaneous orintramuscular injection, or by catherization of lymphatic vessels.

It is considered well-known to those of skill in the art how toformulate a proper composition of a labeled antibody for any of theaforementioned injection routes.

The timing of the administration can vary substantially. The entire dosecan be provided in a single bolus. Alternatively, the dose can beprovided by an extended infusion method or by repeated injectionsadministered over a span of weeks. A preferable interval of time is sixto twelve weeks between radioimmunotherapeutic doses. If low doses areused for radioimmunotherapy, the RIC could be administered at two weekintervals. If the total therapeutic dose is fractionally delivered, itcould be administered over a span of 2 to 4 days. Due to the lower doseinfused, trace-labeled doses can be administered at short intervals; forclinical purposes, one to two week intervals are preferred.

The radiometric dosage to be applied can vary substantially. Lymphomasare known to be radiosensitive tumors. Furthermore, the anti-CD20antibodies appear to have some effect upon lymphomas even whenadministered as unlabeled reagents. There is some evidence that thiseffect is mediated by "apoptosis", a reprogramming of the cellularmetabolism that leads to lysis of the apoptotic cell (78). Forimmunodiagnostic imaging, trace-labeling of the antibody is used,typically 1-20 mg of antibody is labeled with about 1 to 35 mCi ofradioisotope. The dose is somewhat dependent upon the isotope used forimaging; amounts in the higher end of the range, preferably 20 to 30mCi, should be used with ^(99m) Tc and ¹²³ I; amounts in the lower endof the range, preferably 1-10 mCi, should be used with ¹³¹ I and ¹¹¹ In.For imaging purposes about 1 to 30 mg of such trace-labeled antibody isgiven to the subject. For radioimmunotherapeutic purposes, the antibodyis labeled to high specific activity. The specific activity obtaineddepends upon the radioisotope used; for ¹³¹ I, activity is typically 1to 10 mCi/mg. The antibody is administered to the patient in sufficientamount that the whole body dose received is up to 1100 cGy, butpreferably less than or equal to 500 cGy. The amount of antibody,including both labeled and unlabeled antibody, can range from 0.2 to 40mg/kg of patient body weight.

An amount of radioactivity which would provide approximately 500 cGy tothe whole body is estimated to be about 825 mCi of ¹³¹ I. The amounts ofradioactivity to be administered depend, in part, upon the isotopechosen. For therapeutic regimens using ¹³¹ I, 5 to 1500 mCi might beemployed, with preferable amounts being 5 to 800 mCi, 5 to 250 mCi beingmost preferable. For ⁹⁰ Y therapy, 1 to 200 mCi amounts of radioactivityare considered appropriate, with preferable amounts being 1 to 150 mCi,and 1 to 100 mCi being most preferred. The preferred means of estimatingtissue doses from the amount of administered radioactivity is to performan imaging or other pharmacokinetic regimen with a tracer dose, so as toobtain estimates of predicted dosimetry.

A "high-dose" protocol, in the range of 200 to 600 cGy (or higher) tothe whole body, typically requires the support of a bone-marrowreplacement protocol, as the bone-marrow is the tissue which limits theradiation dosage due to toxicity. A preferable dosage is in the range of15 to 150 cGy to the whole body, the most preferable range being 40 to120 cGy. Using such a "low-dose" protocol, toxicity to bone marrow ismuch lower and we have found complete remissions are achieved withoutthe requirement of bone marrow replacement therapies.

Either or both the diagnostic and therapeutic administrations can bepreceded by "pre-doses" of unlabeled antibody. The effects of pre-dosingupon both imaging and therapy have been found to vary from patient topatient. Generally, it is preferable to perform a series of diagnosticimaging administrations, using increasing pre-doses of unlabeledantibody. Then, the pre-dose providing the best ratio of tumor dose towhole body dose is used prior to the administration of theradioimmunotherapeutic dose.

Goldenberg et al. describe radioimmunodiagnostic imaging andradioimmunotherapy of solid tumors (carcinomas) using ananti-carcinoembryonic antigen antibody. Many aspects of the materialsand methods described by them in U.S. Pat. Nos. 4,348,376 and 4,460,559,hereby incorporated in their entirety by reference, can be applied aswell to the present invention, which is directed to the diagnosis andtherapy of lymphoma, a more disseminated tumor. Additional descriptionof methods for estimating the radiometric dose received by a patient areprovided in reference 79.

The present invention also embodies articles of manufacture whichcomprises written material describing the use of an anti-CD20 antibody,or other antibody directed to an antigen associated with cells of the Bcell lineage, in a radioimmunodiagnostic and/or radioimmunotherapeuticprotocol. The written material can be applied directly to a container(such as by applying a label directly to a vial containing theantibody). Alternatively, holding the antibody can be placed in a secondcontainer, such as a box, and the written material, in the form of apackaging insert, can be placed in the second container together withthe first container holding the antibody.

The written portion of the article of manufacture should describeindications for prescribing the antibody. Such indications would bepresentation of lymphoma at any site in the body. The written materialshould further describe that the anti-CD20 antibody, or other antibodydirected to an antigen associated with cells of the B cell lineage, isuseful for the treatment of lymphoma or other neoplasm clonally derivedfrom a cell of B cell lineage, indicated as set forth above. In apreferred embodiment of this aspect of the invention, the writtenmaterial will describe B1, B2, B3, B4 or J5 as the antibody to be usedin the treatment. In a most preferred embodiment, the written materialwill describe that B1 is used in the treatment of lymphoma. In otherpreferred embodiments, the written material will describe that theantibody is labeled with a β⁻ emitting radionuclide, preferably ¹³¹ I₁,¹⁸⁶ Re, ¹⁸⁸ Re or ⁹⁰ Y₁. Still further, it can be described in thewritten material that the appropriate radiometric dose to beadministered for an immunodiagnostic scanning is provided by 1 to 35 mCiof radioisotope, while the appropriate dose for therapeuticadministration should be below 150 cGy to the whole body if bone marrowreplacement support cannot be provided, but can be as high as 600 cGy tothe whole body if bone marrow replacement support is provided. The dosesfor particular isotopes, especially as set forth hereinbelow, might alsobe described.

The written material would preferably be provided in the form requiredby the Food and Drug Administration for a package insert for aprescription drug. The written material would indicate that the antibodywould be prescribed for use in patients having a diagnosis of B celllymphoma and can be administered to patients presenting lymphoma in anysite in the body. The written material would indicate that the antibodyis useful as an initial or secondary treatment or in combination withother treatments. It would further describe that while the antibodydelivers radiation preferntially to tumor sites, sometimes it will beobserved that a normal organ will receive a radiometric dose higher thanthat delivered to the tumor. Principal toxicities would be described as:myelosuppression, perhaps requiring bone marrow or stem cell reinfusionadjunct therapy, and fever, chills and/or hypotension upon infusion,hives and other typical allergic reactions. It would further bedescribed in the written material that when symptoms such as fever orchills or other indications of allergic reactions are observed, HAMAshould be tested. Additional side effects to be described are fatigue,usually mild and of a term of 4 to 6 weeks, and nausea, which is rare,but has been observed.

The written material should also describe that side effects presentingas allergic reactions might be related to the dose rate and can beameliorated by slowing or stopping infusion of the antibody. Also, itshould be described that fever and chills can be treated with Demerol™,Tylenol™, and/or an antihistamine, such as Benadryl™, and thathypotension responds well to fluid administration.

The written material should also describe that delivery of the antibodyis preferably by slow intravenous infusion and might indicate a periodfor the infusion of one to 24 hours. Contraindications of the antibodyare HAMA or previous allergic reaction and pregnancy. Furthermore,precautions should be described in the written material such as that itis recommended that patients be pre-medicated with acetominophen (650mg) and Benadryl™ (50 mg) prior to beginning the infusion. All patientsreceiving ¹³¹ I-antibody should receive 2 drops of saturated potassiumiodide solution (SSKI) three times daily for the period from 24 hoursprior to the administration of the radiolabeled antibody until 14 daysafter the last administration. It should further be noted that serumshould be monitored for HAMA response prior to the first administration,during therapy and follow-up.

The written material should also indicate that general radiologic andnuclear medicine precautions appropriate to the isotope used forlabeling the antibody should be observed.

The following examples of the present invention are provided toillustrate the invention in more detail. The examples are to be taken asillustrative only, without limiting the scope of the invention.

EXAMPLE I LOW-DOSE RADIOIMMUNOTHERAPY OF LYMPHOMA USING B1 ANTIBODY

Methods

Anti-B1 Antibody Preparation and Iodination

The mouse IgG2a monoclonal antibody anti-B1 (anti-CD20) was provided byCoulter Corporation (Coulter Clone™ B1), Hialeah, Fla. It binds to a 35kD cell-surface phosphoprotein expressed by greater than 95 percent ofnormal. B cells isolated from peripheral lymphoid tissues, and bonemarrow and greater than 90 percent of B cell lymphomas (5). It does notbind T cells, granulocytes, monocytes, erythrocytes, hematopoietic stemcells, nor any normal non-hematopoietic tissues (12).

The B1 antibody was isolated from serum-free hybridoma supernatantsproduced in cartridge-type bioreactors and purified by ion exchangechromatography. The resulting preparation was greater than 98 percentpure monomeric IgG, sterile, pyrogen-free, and free of adventitiousviruses.

Radioiodination was performed using the iodogen method (45). Afterpassage through an ion exchange column, which retains free iodine andallows passage of the labeled antibody, greater than 90 percent of ¹³¹ Iactivity was protein-bound by thin layer chromatography. Alternatively,free iodine can be removed using a gel filtration column. The meanspecific activities of trace-labeled and RIT-dose preparations were 0.83and 8.8 mCi per mg, respectively. A rapid direct cell binding assay wasperformed before infusion using a one-hour incubation period to verifypreservation of immunoreactivity as described previously (46).Lyophilized target B cells (Coulter Corporation) were reconstituted indistilled water and diluted in 2 percent bovine serum albumin inphosphate-buffered saline and incubated with radiolabeled antibody underconditions of antigen excess and in the presence or absence of excessunlabeled B1. For trace-labeled preparations, measured direct cellbinding averaged 58 percent, and for RIT dose preparations, 49 percent.These represent minimum estimates of immunoreactivity not extrapolatedto infinite antigen excess (47).

All radiolabeled antibody preparations were sterile-filtered anddetermined to be pyrogen-free by Limulus amebocyte lysate assay prior toinjection. Antibody preparation and administration conformed to anapproved Notice of Claimed Investigational Exemption for a New Drug.

Patient Selection

Adult patients with non-Hodgkin's lymphoma who had relapsed after orfailed to respond to at least one prior chemotherapy regimen wereeligible. Only patients whose tumor tissue was reactive with either B1(by immunoperoxidase staining of cryopreserved tumor biopsies) or withL26 antibody (by staining of paraffin embedded tissue) were eligible. B1and L26 both specifically bind the CD20 antigen (48). An iliac crestbone marrow biopsy was required to show to that less than 25 percent ofthe hematopoietic marrow elements were composed of lymphoma cells. Othereligibility requirements included lack of other treatment for at leastfour weeks, entry absolute granulocyte count greater than 1,500 permicroliter and platelet count greater than 100,000 per microliter,normal hepatic and renal function, lack of other serious illnesses,Kamofsky performance status of at least 60, a life expectancy of atleast 3 months, the presence of measurable disease, and the absence ofserum human anti-mouse antibodies (HAMA). All patients provided writteninformed consent to the protocol which was approved by the InstitutionalReview Board of the University of Michigan.

Radiolabeled Antibody Administration

All patients were hospitalized and first received a trace-labeled dose(approximately 5 mCi, 15 mg) of ¹³¹ I-B1 intravenously over 30 minutes.Biodistribution studies (described below) then followed. To evaluate theeffect of unlabeled antibody predosing on radiolabeled antibodybiodistribution and tumor targeting, a second trace-labeled dose wasgiven approximately one week later which was immediately preceded by a90-minute infusion of 135 mg of unlabeled B1. In some instances, a thirdtrace-labeled dose was given one to two weeks later which was precededby a 90-minute infusion of 685 mg of unlabeled antibody.

At least one week after the last trace-labeled dose, a higherradioactivity level RIT dose was administered. This RIT dose (15 mg) wasgiven with the unlabeled antibody predose which resulted in the highesttumor/whole-body dose ratio in preceding trace-labeled dosebiodistribution studies in that patient. The radioactivity dose (in mCi)administered for RIT was adjusted for each patient so that the patientwould receive a specified whole-body radiation dose (cGy) predicted bythe patient's trace-labeled dose biodistribution results. Sequentialgroups of at least three patients are scheduled to receive escalatingwhole-body doses, starting at 25 cGy and escalating by 10 cGy incrementsuntil a maximal tolerated dose not requiring bone marrow transplantsupport has been defined. Patients were eligible for retreatment after 8weeks if they had not developed a HAMA response, had not experienceddose-limiting toxicity, had stable disease or tumor regression withmeasurable persistent disease, and if their blood counts, hepatic andrenal function, and performance status were in a range that wasoriginally required for protocol entry. Retreatment consisted of atrace-labeled dose (usually with the same unlabeled antibody predoseused for the prior RIT dose) followed one week later by a RIT dose (alsowith the same unlabeled antibody predose) adjusted to deliver the samewhole-body radiation dose delivered by the prior RIT dose.

Diphenhydramine (50 mg) and acetaminophen (650 mg) were given orally aspremedication one hour prior to each infusion. Potassium iodide (SSKI)was given (two drops orally three times daily) beginning the day priorto the first antibody infusion and continuing until 14 days after thelast infusion to inhibit thyroid uptake of radioiodine. Potassiumperchlorate (200 mg three times daily for 7 days) was given in additionto SSKI to patients receiving RIT beginning the day of the RIT infusion.Patients were monitored for alterations in vital signs and for adversereactions every 15 minutes during infusions. After RIT doses, patientswere isolated in lead-shielded rooms until their whole-body radiationlevel was less than 30 mCi by ionization chamber measurements.

Dosimetric, Biodistribution, Pharmacokinetic, and Tumor Imaging Studies

Serial conjugate anterior and posterior whole-body and spot gamma camerascans, as well as NaI scintillation probe whole-body radioactivitycounts, were obtained beginning one hour after trace-labeled doseadministration and then daily for at least 5 days as previouslydescribed (49). Post RIT scanning began after a patient's whole-bodyradioactivity level was less than 30 mCi. Regions of interest outlineswere drawn around normal organs, imaged tumors, and appropriatebackground regions by a computer. Time-activity curves(radioactivity/gram of tissue versus time) corresponding to theseregions were then generated and fit by a least-squares regressionprogram to derive an estimate of cumulative activity. Organ and tumorweights were derived from CT scan volumes when available, otherwisestandard values for reference male or female organ masses were used(50). Dosimetric estimates were then made by the MIRD method (51-54).Blood radioactivity clearance was determined from sequential bloodsamples drawn immediately through 120 hours post infusion and counted bygamma counter. Sera were also obtained for detection of immune complexformation (measured by high-performance liquid chromatography and Clqbinding assays) within two hours following infusion. Urine was alsocollected at designated time intervals after infusion to measure therenal excretion rate.

Gamma scans were interpreted by a single experienced reader and comparedwith prestudy physical examinations, body CT scans, and otherappropriate radiographic studies to determine tumor imaging sensitivity(49).

Toxicity Evaluations

Toxicity was scored according to the National Cancer Institute CommonToxicity Criteria. Complete blood cell and platelet counts were obtainedimmediately after each infusion and then at 2, 4, 24, 72, and 120 hourspost infusion. After discharge from the hospital, blood counts wereobtained weekly for at least 8 weeks. Hepatic enzyme, renal, andelectrolyte studies were performed at least twice during the week afteran infusion and once every two weeks for the first two months afterdischarge. Serum complement levels (C3 and C4) were assayed within 2hours following infusion. Peripheral blood immunophenotyping by flowcytometry was performed before and 24 hours after trace-labeled antibodyinfusions and one to two months post RIT. Direct staining ofFicolliHypaque separated mononuclear cells was performed with B1 andanti-CD19 antibodies for identifying B cells and with anti-CD3antibodies for identifying T cells. Other antibodies used includedanti-CD4, anti-CD8, anti-CD14, anti-CD45, and irrelevantsubclass-matched antibodies.

HAMA responses were assessed from sera obtained prestudy, weekly untiltwo months after the last antibody infusion, and monthly thereafterusing a sandwich enzyme-linked immunosorbent assay described previously(49). Quantitative serum immunoglobulin levels and thyroid functiontests were obtained prestudy, one month post RIT, and then severalmonths post RIT.

Tumor Response Evaluations

Response was assessed during the tracer study interval prior to RIT, 4to 6 weeks post RIT, and every two to three months thereafter. Acomplete remission (CR) was defined as complete disappearance of alldetectable disease for a minimum of 4 weeks, a partial response (PR) asat least a 50 percent reduction in the sum of the products of thelongest perpendicular diameters of all measurable lesions for a minimumof four weeks, and progressive disease (PD) as at least a 25 percentincrease or the appearance of new lesions.

RESULTS

Ten patients entered on this study were initially evaluated and theircharacteristics are shown in Table 1. Half of the patients had low-gradelymphomas while the others had intermediate-grade lymphomas. At entry,three had high tumor burdens (greater than 500 g by CT and physicalexamination), two had low tumor burdens (less than 50 g), and five hadintermediate tumor burdens (50-500 g). The patients were generallyheavily pretreated with chemotherapy (mean number of regimens perpatient=2.7). Half had chemotherapy-resistant disease, as defined by theinability to maintain a response lasting more than one month after thelast administration of chemotherapy.

Gamma camera scans obtained after trace-labeled doses of anti-B1demonstrated distinct tumor imaging of

                                      TABLE 1                                     __________________________________________________________________________    Clinical Characteristics of 10 Patients with B-Cell Lymphoma.                                                                    CHEMOTHERAPY-              PATIENT                                                                             AGE TUMOR   TUMOR   MARROW    PREVIOUS       RESISTANT                  No.   (YR)                                                                              HISTOLOGY*                                                                            BURDEN (g)                                                                            INVOLVEMENT                                                                             THERAPY†                                                                              DISEASE                    __________________________________________________________________________    1     40  DML     >500    +         CHOP, Cyt-E-MTX,                                                              NovACOP-B                                 2     46  FSC      469    +         CVP            +                          3     60  DLC     >500    -         m-BACOD, DHAP,                                                                BEAC + ABMT                               4     42  F & DML >500    -         m-BACOD, BCNU-E-MTX-                                                                         +                                                              Pred, MINE, interferon                    5     56  DLC      236    -         CHOP           +                          6     36  FSC & LC                                                                                60    -         CVP, CHOP, BACOP, XRT                     7     74  DLC      <50    -         CHOP, LEMP, Chlor-Pred,                                                                      +                                                              XRT                                       8     70  DSC      <50    +         VACOP-B, DHAP                             9     59  FML      280    -         Chlor, ProMAC/MOPP,                                                                          +                                                              interferon, chlorambucil,                                                     Cytox-Pred, CEPP, XRT,                                                        DICE                                      10    57  FML      200    -         CHOP, CVP, XRT                            __________________________________________________________________________     *DML denotes diffuse mixed large and smallcell lymphoma; FSC, follicular      smallcleaved-cell lymphoma; DLC, diffuse largecell lymphoma; F & DML,         follicular and diffuse mixed large and smallcell lymphoma; FSC & LC,          follicular smallcleaved-cell lymphoma and follicular largecell lymphoma i     separate lesions; DSC, diffuse smallcleaved-cell lymphoma; and FML,           follicular mixed large and smallcell lymphoma.                                † CHOP denotes cyclophosphamide, doxorubicin, vincristine, and         prednisone; CytE-MTX, cyclophosphamide, etoposide, and methotrexate;          NovACOPB, mitoxantrone, doxorubicin, cyclophosphamide, vincristine,           prednisone, and bleomycin; CVP, cyclophosphamide, vincristine, and            prednisone; mBACOD, methotrexate, bleomycin, doxorubicin,                     cyclophosphamide, vincristine, and dexamethasone; DHAP, dexamethasone,        highdose cytarabine, and cisplatin; BEAC + ABMT, carmustine, etoposide,       cytarabine, and cyclophosphamide, with autologous bone marrow                 transplantation; BCNUE-MTX-Pred, carmustine, etoposide, methotrexate, and     prednisone; MINE, methotrexate, ifosfamide, mitoxantrone, and etoposide;      BACOP, bleomycin, doxorubicin, cyclophosphamide, vincristine, and             prednisone; XRT, external beam irradiation; LEMP, lomustine, etoposide,       methotrexate, and prednisone; ChlorPred, chlorambucil and prednisone;         VACOPB, etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone     and bleomycin; ProMACE/MOPP, methotrexate, doxorubicin, cyclophosphamide,     etoposide, mechlorethamine, vincristine, procarbazine, and prednisone;        CytoxPred, cyclophosphamide and prednisone; CEPP, cyclophosphamide,           etoposide, procarbazine, and prednisone; and DICE, dexamethasone,             ifosfamide, cisplatin, and etoposide.                                    

all known disease sites larger than 2 cm in all patients (30 of 30 knownsites, range=1 to 9 per patient). Lesions from 1 cm to 15 cm in diametercould be detected, including intrasplenic tumors (FIG. 1).

Unlabeled B1 predosing was performed to assess the effect of suchpre-dosing on the distribution of subsequently administered unlabeledantibody to tumors through partial or complete presaturation ofnon-specific binding sites and/or reservoirs of non-malignant B cells(especially those in the spleen). Predosing consistently prolonged bloodand whole-body clearance of radioisotope compared to clearance oftrace-labeled antibody without predosing, but its effect on radiolabeledantibody tumor targeting relative to normal tissues was variable. Ofeight patients who received a 135 mg unlabeled antibody predose, two hada greater than 20 percent improvement in a tumor/whole-body dose ratiocompared to a prior trace-labeled dose given without predosing and threehad no significant improvement. Three were unassessable due to eithertechnical difficulties in dose assessment associated with the proximityof organs involved in excretion of free iodine or due to tumor volumedecreases after trace-labeled antibody infusion. Two of two patientsgiven subsequent trace-labeled doses preceded by a 685-mg unlabeledpredose were also not assessable because of tumor responses, that is,decreases in tumor volumes, occurring after these infusions.

Table 2 shows that calculated radiation doses delivered to tumors byunlabeled antibody exceeded those to any normal organ in all but two ofseven assessable patients. Up to 24.1 cGy per mCi (mean=10.6±2.76) couldbe delivered to tumor. Features unique to the two patients with poorertargeting which could account for suboptimal outcome were grosssplenomegaly (750 g) in Patient 1 (the spleen could act as an "antigenicsink" for the labeled antibody) and a high degree of sclerosis in thetumor in Patient 5 (which might limit access of antibody to tumorcells).

Nine patients received RIT doses and were evaluable for response andtoxicity (Table 3). One patient did not receive an RIT dose because ofrapid tumor progression and deterioration of physiologic status duringtracer studies to the point of making the patient ineligible forprotocol treatment. Four patients were treated twice (about two monthsbetween treatments). An estimated 25 to 45 cGy were delivered per doseto the whole body using 34 to 66 mCi per dose. Six of the nine patientshad significant tumor responses, including four complete remissions(CRs) and two partial responses. Responses were observed in patientswith extensive and/or bulky and chemotherapy-resistant disease (e.g.FIG. 2A and 2C). Three patients had responses (e.g. FIG. 2B and 2D)which began after trace-labeled doses even before RIT doses were given.All four patients

                                      TABLE 2                                     __________________________________________________________________________    Doses of Radiation Delivered by [.sup.131 I]Anti-B1 Antibody to Various       Sites.                                                                                        SITE (cGy/mCi)*                                               PATIENT                                                                             PRETREATMENT    WHOLE                                                   No.   DOSE (g)  TUMOR†                                                                       BODY BLOOD                                                                              KIDNEYS                                                                             LIVER                                                                              LUNGS                                                                              SPLEEN                        __________________________________________________________________________    1     135        2.68 0.48 2.02 3.36  2.07 1.74 3.75                          2     135        8.01 0.74 4.84 4.49  2.62 2.84 7.42‡              4      0         8.77 0.55 3.03 6.14  2.45 2.33 2.01                          5      0         3.72 0.88 4.48 6.24  2.49 2.38 7.31                          6     135       24.1  0.76 3.87 4.43  2.39 2.23 4.49                          9      0        12.1  0.74 4.44 4.36  2.23 1.72 8.12                          10    135       14.6  0.73 5.76 7.24  3.12 4.38 3.47                          Mean ±       10.6 ±                                                                           0.69 ±                                                                          4.06 ±                                                                          5.18 ±                                                                           2.48 ±                                                                          2.52 ±                                                                          5.22 ±                     SE               2.76 0.05 0.46 0.52  0.13 0.34 0.89                          __________________________________________________________________________     *Values shown reflect the tracelabeled doses resulting in the highest         delivered dose to tumor in each patient.                                      † Values shown are the maximaldoses delivered to any of a patient'     tumors.                                                                       ‡ CT scans showed multiple lowattenuation lesions in the splee     that were consistent with involvement by lymphoma.                       

                                      TABLE 3                                     __________________________________________________________________________    Responses to Radioimmunotherapy.                                                    WHOLE-BODY                                                                    DOSE AND   ANTIBODY DOSE                                                      ACTIVITY   THERAPEU-                                                                              TOTAL                      PERIOD WITH-             PATIENT                                                                             ADMINISTERED                                                                             TIC DOSE DOSE*                                                                              HEMATOLOGIC                                                                              TUMOR      OUT DISEASE              No.   cGy   mCi  mg            TOXICITY   RESPONSE   mo                       __________________________________________________________________________    1     25    66   150      315  Grade 1    Disease progression                 2     25    45    15      180  None       Partial response                                                                          5                             25    34    15       30  Grade 1                                        3     No    Not            15                                                       treated                                                                             treated                                                           4     25    57    15      180  Grade 1    Complete remission†                                                                8                             25    37    15       30  Grade 2                                        5     35    38    15       30  Grade 1    Disease progression                 6     35    40   700      1565 Grade 1    Complete remission‡                                                           ≧11               7     35    41   150      315  None       Disease progression                       35    40    15       30  None                                           8     35    40   150      315  Grade 2    Complete remission§                                                                 ≧9                9     45    44   150      315  None       Partial response¶                                                              ≧2                      45    44   150      300  Not eavl-  Mixed response**                                                   uated∥                                10    45    61   150      1015 None       Complete remission                                                                       ≧8                __________________________________________________________________________     *Includes tracer doses.                                                       † A remnant softtissue abnormality was seen in the area of previou     tumor involvement on CT scanning afte the first radioimmunotherapeutic        dose; it did not substantively change after the second                        radioimmunotherapeutic dose. This remnant abnormality was believed to be      scar tissue, because when the patient relapsed after eight months in the      right paracaval region and conventional external beam irradiation to the      abdomen was given, no change occurred.                                        ‡ Complete remission was induced with tracer doses.                § This was a probable complete remission in a patient with               mediastinal and axillary adenopathy and bone marrow involvement before        therapy and a solitary, small, nonaratrabecular lymphoid aggregate found      in followup biopsy speciimens of iliaccrest bone marrow.                      ¶ Partial response was induced with tracer doses and further        response by radioimmunotherapeutic doses.                                     ∥ Although grade 3 thrombocytopenia was observed one month after     radioimmunotherapy, this patient had rapidly progressing disease in           previously involved and uninvolved sites, necessitating intervention with     other treatment. Because no bone marrow biopsy was performed before the       managing physician began therapy, no distinctino could be made between        peripheral bloodplatelet destruction, progressive infiltration of marrow      by lymphoma, and marrow toxicity.                                             **See the Results section for details.                                   

with CRs achieved this status after only one RIT dose. Second RIT dosesresulted in a mixed response in one patient (definite regression in sometumors and progression in others), no further disease response in two,and no change in a residual radiographic abnormality in one. One CRlasted 8 months, and three CRs continue progression-free for 8+ to 11+months. Minimal toxicity was observed in all of the fully evaluablepatients. Most had either reversible Grade I myelosuppression(leukopenia and/or thrombocytopenia) occurring 4 to 7 weeks post RIT orno toxicity. One patient had a mild rigor and fever during an RIT doseinfusion.

Peripheral blood flow cytometry revealed that CD20-positive B cellsconstituted 2 to 20 percent of circulating mononuclear cells at baselinein our patients. Most had decreases in the percentage of CD20-positivecells 24 hours after tracer infusions with three patients showingcomplete depletion of these cells. All patients recovered their CD20cell counts to close to baseline generally one to three months after RITand did not show any evidence of increased rates of infection. Nosignificant changes in circulating CD3-positive T cells were observed.Also, no significant changes in serum immunoglobulin levels have beenseen with continued follow-up, including five patients who had lowlevels prestudy. Only two patients developed HAMA responses 53 and 81days after the first trace-labeled antibody infusion. No instances ofhypothyroidism induced by thyroid irradiation have yet been observed.

DISCUSSION

A striking proportion (four of six) of our observed responses have beencomplete and durable. This, together with the observation that responsescould be achieved with a relatively short course of treatment inpatients with bulky and/or extensive disease and chemotherapy-resistantdisease, attests to the utility of this treatment. Although the ultimatelength of complete remission in some of our patients has yet to bedetermined, this therapy offers, at the very least, excellent palliationof disease in view of its lack of toxicity and the potential forrepeated treatments if relapse should occur.

Several factors may account for the excellent results obtained so far inour current trial and for why they appear better than those of othertrials. First, anti-B1 antibody appears to be a superior tumor targetingagent. Indeed, the ability to clearly image all tumors larger than 2 cmand even tumor lesions within the spleen (an organ rich in normal Bcells) suggests a potential diagnostic role for radiolabeled anti-B1.These results compare favorably with those using the LL2 antibody (63),are superior to those we previously obtained with MB-1 (56) and to thosereported with the anti-CD21 antibody OKB7 (66). Also, our estimates ofthe radiation dose delivered to tumor in cGy per mCi by ¹³¹ I-B1(10.6±2.76) appears to be at least double that reported for otherradiolabeled B cell antibodies (56,63,64,66). This may be, in part,because of the high degree of specificity of B1 for B cells and its lackof crossreactivity with other cells. Also, in contrast to the antigenstargeted by other studied antibodies, the CD20 antigen does not modulate(i.e., disappear from the cell surface via cytosolic internalization orcell surface membrane shedding) after antibody binding (12). Sinceinternalization of radiolabeled antibody may result in dehalogenation ofantibody and subsequent release of free iodine from the cell (67), theabsence of such a mechanism may result in prolonged retention of intactradiolabeled antibody by the targeted cell. Notably, better tumortargeting appeared to translate into improved tumor responses in ourpatients. Those patients with relatively poorer targeting did not, ingeneral, respond to treatment. This may also indicate that the tumorresponses observed were more likely due to antibody-targeted radiationrather than simply whole-body irradiation.

In one patient with gross splenomegaly (Patient 1), significantimprovement with antibody predosing was seen. When no predose was givento this patient, radioactivity localized predominantly to the spleen andno tumor sites were detectable, but with a 135-mg predose, splenicuptake of radioisotope was much reduced and the patient's multiple tumorsites became detectable. This supports our hypothesis that unlabeledantibody predosing may help radiolabeled antibody to bypass an antigenicsink (such as the spleen) and allow its better access to tumor sitesthrough competitive binding mechanisms between unlabeled and labeledantibody.

Another factor potentially accounting for our results is the lowradiation dose-rate associated with this form of delivery of radiationto targeted tumor cells. Animal model data have suggested that lowdose-rate irradiation may, in fact, be more therapeutically effectivethan instantaneous irradiation fractionally delivered by conventionalexternal beam (68-70), but the radiobiologic basis for this is stillunclear. Also, recent observations have indicated that low dose-rateirradiation can induce apoptosis in lymphoid cell lines and thatantibody binding to cells (including B1 binding) can synergize with thismode of irradiation to induce this effect (71,72).

The antibody moiety of the ¹³¹ I-B1 conjugate may also be partlyresponsible for antitumor effects. B1 is capable of inducingantibody-dependent cellular cytolysis (73) and complement-dependentcytolysis (20), probably because its Fc portion is of the IgG2asubclass. Also, B1 can directly induce apoptosis in certain human B celllymphoma cell lines. We have recently studied B1 antitumor effects invivo in a human B cell xenograft nude mouse model and have found thatunder certain conditions unlabeled B1 can have comparable inhibitoryeffects on tumor growth to ¹³¹ I-labeled anti-B1 (73). Our observationof tumor responses during tracer studies in our clinical trial alsosuggests an antitumor role of the B1 antibody moiety. The relativelyhigh dose of unlabeled antibody administered in some patients may havecontributed to these responses. Indeed, in two of three instances inwhich a response occurred during tracer studies, the response was onlyseen after the largest dose of antibody (700 mg) was administered(Patients 6 and 10). However, in these cases and those in which aresponse appeared to occur only after an RIT dose, a targeted radiationeffect is also likely, especially since targeting of radioisotope wasfound to be so high in these cases and could result in the delivery totumor of up to 120 cGy per tracer dose (Table 2). Finally, it iscertainly possible that at least six different anti-tumor mechanismsdiscussed above can be working in concert either additively orsynergistically in this treatment including 1) antibody-targetedradiation, 2) low dose-rate irradiation and its incompletely understoodeffects, 3) whole-body irradiation, 4) antibody-dependent cellularcytolysis, 5) complement-dependent cytolysis, 6) and antibody-inducedapoptosis.

EXAMPLE II ADDITIONAL PATIENTS TREATED BY ¹³¹ I B1 RADIOIMMUNOTHERAPY

Twelve additional patients were treated essentially as described inExample I, bringing the total number of patients to 22. The cumulativeresults of all 22 patients are summarized as follows:

All received between 1 and 3 trace-labeled doses intravenously (5 mCilabelling 15 mg of B1 antibody), spaced at weekly intervals.Trace-labeled doses were immediately preceded by either no pretreatment,pretreatment with 135 mg and pretreatement with 685 mg of unlabeledantibody. 16 of the patients received additional radioimmunotherapeuticdoses, ranging from 34 to 93 mCi, preceded by that dose of unlabeledantibody that resulted in the best tumor imaging in the tracer study.Six patients were unable to receive radioimmunotherapeutic doses dueeither to disease progression resulting in physiologic deterioration(n=3) or due to development of a HAMA response (n=3). Of the 22patients, 15 exhibited a tumor response (CR or PR). Of the 16 patientsreceiving a radioimmunotherapeutic dose, 13 exhibited CR or PR. Eight ofthe patients who received a radioimmunotherapeutic dose exhibited CR. Ofthese 8 patients with CR, 2 have relapsed (8 and 13 months postRIT) andthe remaining 6 have remained disease free 16, 13, 8, 6, 3 and 2 monthspost-RIT, respectively. Tumor responses began during the tracer studiesin 11 of the 22 patients, including 9 of the RIT patients, but thegreatest proportion of the response, and the fastest rate of change,occurred following RIT. Toxicity has been minimal; maximum hematologictoxicity=grade 3 has been seen in only 3 patients, and that of shortduration. The whole body dose range administered in patients to date hasbeen between 25 and 65 cGy. The minimal toxicity observed indicates thatescalation of doses is appropriate.

EXAMPLE III ⁹⁰ Y RADIOIMMUNOCONJUGATES IN RADIOIMMUNOTHERAPY OF LYMPHOMA

General Considerations in Choosing Radioisotopes

For radioimmunoscintigraphy the radioisotopes of choice arecharacterized by relatively low energy gamma emissions with a physicalhalf-life in the range of 6 hours to 8 days. A gamma emitter withprinciple emissions in the 0.1 to 0.2 Mev range is most ideal forscintigraphy, because the detection equipment is built with a focus on^(99m) Technetium which accounts for most of the Nuclear Medicineimaging procedures. The thickness of the detection device and collimatorrequired for imaging with higher energy gamma emitters contribute to thefuzzy images obtained with high energy gamma emitters such as ¹³¹Iodine. An intact monoclonal antibody requires a period of hours to daysto localize in tumor and a period of days for the blood pool and normalorgan background to clear. Therefore, radioisotopes with very shorthalf-lives are not very useful. Thus the initial dose in mCi must bevery large for a short-lived radioisotope to have sufficient activityremaining at optimal imaging times. Radioiodines have been usedextensively, but they suffer extensively from dehalogenation, especiallyupon internalizaion of the RIC into the target cell, and the lack ofradioiodine with ideal characteristics (¹²³ Iodine is probably theclosest, but it suffers from great expense, uncertain supply of proteiniodination grade material, and short half-life). Of the readilyavailable radioisotopes for radioimmunoscintigraphy with an intactmonoclonal antibody ¹¹¹ Indium is the current radioisotope of choice.

Gamma emitters are not suitable for radioimmunotherapy. α, β⁻, and augerelectron emitting radioisotopes have been proposed forradioimmunotherapeutic applications. Although α-emitting radioisotopesare an area of great research interest, there are no readily availableα-emitting radioisotopes for which chelation chemistry has beendeveloped that have isotopic half-life characteristics that match thepharmacokinetics of IgG monoclonal antibodies. The auger electronemitter ¹²⁵ I has been used for therapy purposes, but it suffers fromdehalogenation and a long isotopic half-life (60 days). Several β⁻-emitting isotopes appear to have promising characteristics forradioimmunotherapy. ¹⁸⁶ Re and ¹⁸⁸ Re are promising β⁻ -emittingradioisotopes that are under investigation, but theirradioimmunoscintigraphy partner is suited for IgG fragments (Fab' &F(ab')₂), not IgG because of its short 6 hour half-life. A good β⁻-emitting isotope for radioimmunotherapy using intact IgG is ⁹⁰ Y. It isa pure β⁻ -emitting isotope, so the unnecessary exposure by penetratinggamma emissions to hospital staff is minimized. This unfortunately doesnot allow ⁹⁰ Y to be used for radioimmunoscintigraphy for the purpose ofdeveloping predictive dosimetry with a sub-therapeutic dose of ⁹⁰ Ylabeled antibody. The half-life of ⁹⁰ Y (2.6 days) is long enough that asignificant percentage of the radiation dose will be delivered aftertumor localization of the radiolabeled antibody has occurred and itshalf-life is short enough that the isotope will have decayed 97% in 13days, so that hematological and immune system rescue via an autologousbone marrow transplant can be used in a reasonable time frame. A ⁹⁰ Yproduct suitable for high specific activity radiolabeling of chelatedantibodies is available from the Amersham Corporation.

Because no yttrium isotope with characteristics forradioimmunoscintigraphy is readily available, ¹¹¹ In radiolabeledB1-MX-DTPA is used for radioimmunoscintigraphy and the dosimetry thatwould be obtained if ⁹⁰ Y were the radiolabel is estimated. ⁹⁰ Yradiolabeled B1-MX-DTPA is then used for radioimmunotherapy. Weanticipate that there will be some inaccuracy in estimating thedosimetry of the ⁹⁰ Y to normal organs, especially the bone, because ofthe differing pharmacokinetic characteristics of the element iodine(which behaves somewhat like iron and is coordinated by the ironcarrying protein transferrin) and yttrium (which behaves somewhat likecalcium and concentrates in the mineral bone), but we expect that theeffect of these differences in behaviors of these elements will beminimized in the dosimetry estimates, because bone marrow is the organin which the dosimetry estimate will be most affected and autologousbone marrow transplantation can supplement the radioimmunotherapyregimen.

Dose escalation of ⁹⁰ Y labeled B1 can be performed in a cautiousprogression to minimize the chances of irreversible toxicities. Thereproducibility of the correlation between the dosimetry predicted from¹¹¹ In labeled B1 and toxicity and the effect of the ⁹⁰ Y labeled B1upon tumor tissue is an important consideration in use of ⁹⁰ Y labeledantibodies for therapy of cancers.

Characterization of B1 Antigen and Anti-B1 Antibody

B1 antigen is expressed on all B cell cancers except for myelomas. B1antigen is absent from resting or activated T cells, erythrocytes,monocytes, Null cells, and granulocytes. B1 positive B cells occur inlymph nodes, bone marrow, spleen, and circulation. The results intransplants of autologous bone marrow purged with B and complement innon-Hodgkin's B cell lymphoma patients indicated that the B1 antigen isnot expressed on the pre-B stem cell as there is normal reconstitutionof the B cell population. Recent studies of the B1 antigen (CD20)indicate that B cells are the only cell type that express the mRNA forCD20 antigen.

We have studied tissue/organ specific binding of B1 antibody beyondnonspecific scattered binding to tissue where nonspecific controlantibodies also bind. The results of these studies are shown in Tables 4through 6.

By virtue of its similar distribution in the B cell lineage, an antibodydirected against the CD19 antigen is expected to be useful in the samemanner as the B1 antibody. This is especially the case when ⁹⁰ Y or ¹⁸⁶Re is the radioisotope used, since loss of the isotope uponinternalization is not the problem that it is when a radiohalogen isused to label the antibody.

                  TABLE 4                                                         ______________________________________                                        B1 Reactivity with Frozen Normal Human Tissues                                Determined by Avidin-Biotin Immunoperoxidase Staining                                      Reference #6771599                                               Frozen Adult Tissues                                                                       Number Positive/Number Processed                                 ______________________________________                                        Adrenal      0/4                                                              Appendix     0/3                                                              Breast       0/1                                                              Cervix       0/2                                                              Colon          0/4 A                                                          Esophagus    0/3                                                              Fallopian Tube                                                                             0/1                                                              Heart        0/7                                                              Intestine    0/2                                                              Kidney       0/4                                                              Liver        0/3                                                              Lung         0/5                                                              Lymph Node   2/4                                                              Nasal Polyp  0/1                                                              Ovary        0/4                                                              Pancreas      0/2 B                                                           Prostate     0/9                                                              Spleen       5/6                                                              Stomach      0/7                                                              Thymus       1/2                                                              Throid       0/6                                                              Tonsil       3/3                                                              Trachea      0/3                                                              Uterus       0/2                                                              ______________________________________                                         Key:                                                                          (A): scattered glandular nonspecific artifact                                 (B): scattered nonspecific activity in islets of Langerhans cells        

                  TABLE 5                                                         ______________________________________                                        B1 Reactivity with Frozen Normal Fetal Tissues                                Determined by Avidin-Biotin Immunoperoxidase Staining                                      Reference #6771599                                               Frozen Fetal Tissues                                                                       Number Positive/Number Processed                                 ______________________________________                                        Adrenal      0/1                                                              Brain        0/1                                                              Colon        0/2                                                              Heart        0/6                                                              Kidney       0/6                                                              Kideny       0/6                                                              Liver        0/6                                                              Lung         0/6                                                              Pancreas     0/1                                                              Small Intestine                                                                              0/4 A                                                          Smooth Muschle                                                                             0/1                                                              Spleen       5/5                                                              Stomach      0/3                                                              Thymus       0/1                                                              Umbilical Cord                                                                             0/3                                                              ______________________________________                                         Key:                                                                          (A): scattered glandular nonspecific artifact                            

                  TABLE 6                                                         ______________________________________                                        B1 Reactivity with Frozen Lymphoma Tissues Determined by                      Avidin-Biotin Immunoperoxidase Staining                                       Frozen        Reference #6771599                                              Lymphoma Tissues                                                                            Number Positive/Number Processed                                ______________________________________                                        B-Cell        1/1                                                             Hodgkins      0/1                                                             T-Cell        0/1                                                             ______________________________________                                    

We have observed no change in the specificity of B1 monoclonal antibodyafter conjugation with isothiocyanatobenzyl-methyl-DTPA and metalationwith indium using standard conditions. Potential changes in thespecificity of the B1 monoclonal antibody after labeling were addressedby comparing the immunohistochemical tissue specificity andimmunofluorescence (flow cytometric and fluorescence microcopy) patternsof metalated (coordination complex with the stable isotope of In)B1-MX-DTPA versus untreated B1 monoclonal antibody (both the lot B1 thatwas parent to the B1-MX-DTPA and our QC backlot of B1). For this study0.5 mL of nonradioactive I_(n) at 0.2 ug/mL in 0.04M HCl was added to0.5 mL of 0.25M sodium acetate (metal free). B1-MX-DTPA (2 mg) was addedto the neutralized In solution and incubated for 20 min before adding0.25 mL of 0.005M calcium EDTA. The In-B1-MX-DTPA sample was thendiluted in injectable saline.

For immunofluorescence analysis, the In-B1-MX-DTPA was tested side byside with its parent lot of B1 at doses of 20, 5, and 1.25 microgramsantibody per tube of human blood. The population of cells in normalhuman blood that bound B1 were detected using fluoresceinisothiocyanatelabeled goat anti-murine immunoglobulin (GAM-FITC). The whole bloodsamples examined by fluorescence microscopy showed no significantdifference in the percentage of positive cells (3 to 5% of lymphocytes)and negative cells (0% of monocytes or granulocytes) or the intensity ofstaining (mean fluorescence channel of positive cells, 99 to 110 by flowcytometry).

To test that no new population of cells is recognized by theIn-B1-MX-DTPA we performed a dual fluorescence labeling experiment usingphycoerythrin conjugated B1 (PE-Bt) and fluorecein-isothiocyanatelabeled goat anti-murine immunoglobulin (GAM-FITC). Whole blood wasreacted with an equimolar mixture of the PE-B1 and the In-B1-MX-DTPAparent lot. The cells were then washed of excess unbound murine antibodyand reacted with GAM-FITC. In this experiment we would expect thefluorescent cells to fluoresce red (PE-B1) and green (GAM-FITC) if thePE-B1 and In-B1-MX-DTPA or parent lot B1 bind to the same cells. Anycells that reacted only with In-B1-MX-DTPA but not with PE-B1 wouldfluoresce "green" only and thus the conjugated B1 would be considered"damaged" since PE-B1 should bind to all cells bearing B1 antigen. Thevast majority of the blood cells were negative (R-G-,94 to 99% ofcells). Five percent of the cells that were examined by microscopy and1% of cells analyzed by flow cytometric analysis fluoresced both red(PE-B1) and green (GAM-FITC), indicating that B1 and In-B1-MX-DTPArecognize the same population of blood cells as PE-B1. The exception wasthat 1% of the cells examined microscopically appeared positive forPE-B1, but not GAM-FITC (R+G-) for the parent B1 lot. These resultsindicate that indium labeled B1-MX-DTPA and B1 parent lot possesses thesame specificity for circulating blood cells.

Another test of specificity of the metalated-B1-MX-DTPA was to study theimmunohistochemical staining patterns of QC backlot, B1 parent lot, andIn-B1-MX-DTPA using sections of frozen normal (not neoplastic) humantissue (both B cell negative tissues and B cell positive tissues). Nosignificant differences in staining patterns of these three preparationsof B1 are obvious upon examination of the slides. In normal tissue B1 isexclusively a membrane bound antigen. These tissue sections wereconsidered positive if the outer cell membranes of any cells in theentire tissue section stain positive. Cytosolic binding was consideredto be nonspecific in nature. Four adult control tissues (liver,prostate, heart, and uterus) and nine control fetal tissues (intestine,lung, liver, kidney, adrenal, heart, brain, thymus, and colon) wereexamined that should have been negative; all were negative with thethree B1 preparations tested, except for some cytosolic backgroundbinding. Most of the samples of the tissues that should be positive(tonsil, lymph node, and spleen) showed membrane staining typical of B1positive tissues with the three B1 preparations tested. We conclude thatthere is no change in the tissue specificity of B1 that is detectable bythese methods when it is conjugated to ITC-benzyl-DTPA and metalated(labeled) with In.

In non-cancerous tissue B1 appears exclusively as a membrane boundantigen. Tissue sections were considered positive if the outer cellmembranes of any cells in the entire tissue section stain positive.Cytosolic binding of B1 was considered to be nonspecific in nature. Fouradult control tissues (liver, prostate, heart, and uterus) and ninecontrol fetal tissues (intestine, lung, liver, kidney, adrenal, heart,brain, thymus, and colon) were examined that should have been negative,all were negative with the B1 preparations tested, except for somecytosolic background binding. Most of the samples of the tissues thatshould be positive (tonsil, lymph node, and spleen) showed membranestaining typical of B1 positive tissues with the B1 preparations tested.

Labeling of Antibody

We have found that B1 can be successfully conjugated with MX-DTPA andradiolabeled with ¹¹¹ In or ⁹⁰ Y (see below). By this radiolabelingmethod, the antigen reactive fraction for B1-MX-DTPA was 71±8% (S.D.)for 27 determinations.

B1 is radiolabeled with ¹¹¹ In and ⁹⁰ Y, using a chelation labelingmethod originally developed at the National Cancer Institute and refinedby Coulter Immunology.1-P-Isothiocyanato-benzyl-methyl-diethylenetriaminepentaacetic acid(ITC-MX) is used as the chelator. The synthesis of ITC-MX is a 6 stepsynthesis carried out by the Organic Chemistry Department of CoulterImmunology Division.

A diagrammatic representation of this synthesis is presented in FIG. 3.This synthetic route is a modification of the procedure reported byBrechbiel et al (86) for the synthesis of1-p-Isothiocyanato-benzyl-methyl-diethylenetriaminepentaacetic acid(ITC-benzylDTPA). The starting material, 1-p-nitro-L-phenylalaninemonohydrate is obtained from commercial sources and characterized as toidentify and purity by melting point (decomposition), infraredspectroscopy, nuclear magnetic resonance, and analytical reversed phasehigh pressure liquid chromatography. The products are checked foridentity and purity at each step by methodologies that include HPLC,infrared spectroscopy, nuclear magnetic resonance, melting point,thin-layer chromatography, fast atom bombardment mass spectroscopy andelemental analysis. The overall yield of the synthesis is quite good atabout 17% (considering that it is a 6 step synthesis). The final productis filtered through a 0.22 micron sterile filter and aliquoted intosterile tubes using aseptic technique.

The aliquots of ITC-benzyl-MX are stored at -80° C. until use forconjugation with monoclonal antibody. The ITC-benzyl-MX has been shownto be stable for at least 2 years under these storage conditions. Thisfinal product is analyzed by HPLC and functional tests for purity,stability, and performance. The LAL assay for endotoxins indicates thatthe endotoxin concentration of this final product is less than 0.01endotoxin units per mg of ITC-benzyl-MX.

Selection of Patients

Patient eligibility is restricted in our study to histologically or flowcytometric analysis confirmed B1 positive non-Hodgkin's lymphoma whichhas relapsed from conventional primary and salvage regimens. Due to thesomewhat experimental nature of the therapy, patient eligibility is morerestricted than we expect it to ultimately be. At the present time,patients must have a chronological age greater than 18 years. Patientsmust have bone marrow function that qualifies them for an autologousbone marrow transplant. Patients must be of reasonable health other thantheir lymphoma disease without any other malignancies, no uncontrolledviral or fungal infections, be HIV negative and have an expectedsurvival of more than 2 months. At least three weeks must have elapsedsince any prior therapy or surgery. Patients must have reasonable endorgan function and hematopoiesis, including no clinically significantcardiac or pulmonary symptomology. Patients will be excluded for whomthe previously received dose of radiation therapy is so great thatradioimmunotherapy might exceed organ tolerances. Patients must becapable of and give informed consent before entering the study. Patientswith known prior exposure or hypersensitivity to murine proteins or bonemarrow transplant will be excluded. Pregnant and nursing women areexcluded from the study. Patients in whom there is a failure todemonstrate localization of ¹¹¹ In labeled B1 in at least 50% of theknown tumor sites will not receive a dose of ⁹⁰ Y B1.

Composition of the Drug

a. Contents of Vial 1 (10 mL) and Vial 2 (3 mL)

1. Mouse IgG2a Vial 1 4-6 mg/mL Vial 2 35-45 mg/mL

2. Potassium Phosphate 1.7 mg/mL±5%

3. Sodium Chloride 8.5 mg/mL±5%

4. Maltose 100 mg/mL±10%

Final Product:

Coulter Clone® B1 is a clear colorless liquid in a properly labeledglass vial stoppered with a gray silicone coated butyl rubber stopperand capped with an aluminum crimp seal.

b. Contents of Vial 3 (1 mL) (B1-MX-DTPA)

1. Isothiocyanato-benzyl-methylDTPA-Mouse IgG2a 1.8-2.4 mg/mL

2. Sodium Acetate 0.05M+5%

3. Sodium Chloride 8.5 mg/mL±5%

4. Maltose 100 mg/mL±10%

Final Product:

COULTER CLONE® B1 conjugated toIsothiocyanato-benzyl-methyldiethylenetriaminepenta acetic acid is aclear colorless liquid in a properly labeled polypropylene vialstoppered with a gray teflon coated butyl rubber stopper and capped withan aluminum crimp seal.

c. Contents of Vial 4 (0.5 mL) (Acetate buffer)

1. Sodium Acetate 0.25M+5%

Final Product:

Sterile, non-pyrogenic aqueous 0.25M sodium acetate in a properlylabeled polypropylene vial stoppered with a gray teflon coated butylrubber stopper and capped with an aluminum crimp seal.

d. Contents of Vial 5 (1.0 mL) (Quenching Reagent)

1. EDTA 0.005M±5%

2. Sodium Chloride 9 mg/mL±5%

Sterile, non-pyrogenic 0.005M ethylenediamine- tetra acetate (EDTA orVersenate) in 0.9% NaCl in a properly labeled polypropylene vialstoppered with a gray teflon coated butyl rubber stopper and capped withan aluminum crimp seal.

e. Radiolabeling Agents

1. ¹¹¹ In in 0.04N HCl (purchased as a separate component)

¹¹¹ In as cation 10 mCi/mL at assay date.

Final Preparation:

Sterile, non-pyrogenic ¹¹¹ In in 0.04N hydrochloric acid(Medi+Physics/Amersham Corporation product INS.1PA, or equivalent).

2. ⁹⁰ Y in 0.04N HCl (purchased as a separate component) ⁹⁰ Y as cation20 mCi/mL at assay date

Final Preparation:

Sterile, non-pyrogenic ⁹⁰ Y in 0.04N HCl (Medi+Physics/AmershamCorporation product YAS.4P, or equivalent).

The ¹¹¹ In-B1-MX-DTPA and ⁹⁰ Y-B1-MX-DTPA will be diluted withinjectable 0.9% saline containing 5% human albumin (Albuminar-25, ArmourPharmaceutical Company, Kankakee, Ill., Buminate 25%, Baxter HealthcareCorporation, Hyland Division, Glendale, Calif., or equivalent).

Dosimetry

Since dosimetry is required for ⁹⁰ Y, and quantitative imaging of theBremsstahlung is extremely poor, all organ dosimetry (except the bloodfor which direct ⁹⁰ Y data will be obtained) will be derived from ¹¹¹In-RIC images under the assumption that the ¹¹¹ In and ⁹⁰ Y-radiolabeledantibody posses identical pharmacokinetics.

Dosimetry protocol

1. Administer to the patient an imaging dose of ¹¹¹ Inradioimmunoconjugate. An ¹¹¹ In standard will be placed at the level ofthe patient head for determination of the sensitivity of the camera.

2. Patient alignment relative to the table will be moted and reproducedon each imaging session.

3. Obtain anterior/posterior planar images on the whole body camera atthe time points: day 1, day 2 and day 3 setting two 10% windows centeredat the 245 keV and 171 keV emission energies of ¹¹¹ In. An additionalγ-camera imaging session will be performed on day 7 following treatmentwith ⁹⁰ Y and the results will be analyzed for the ¹¹¹ In, and ifpossible ⁹⁰ Y, distribution.

4. Draw region of interest (ROI) around tumor regions, heart, liver,spleen, lung and muscle (background). ROI will be correlated with normalorgans and known tumor volumes from CT scans.

5. Take geometric mean of total pixel count divided by number of pixelsin ROI from opposed views, so that one has a value for the averagecpm/pixel for each organ. This value is almost independent of the depthof the source within the patient, but is dependent on the patientthickness. Patient separation will be measured at head, neck, chest,abdomen, hips and legs.

6. The geometric mean of the cpm/pixel from each ROI will be convertedto an activity of ¹¹¹ In following calibration of the machine using aset of standard specific activity sources of different volume measuredwithin a water phantom for varying water depths in the tank. Theintention of this study is to obtain a relation between mean cpm/pixeland μci/g for ¹¹¹ In, as well as a set of attenuation values for severalwater depths.

7. Obtain the average cpm/pixel for the whole body derived rom images ofthe head, chest, abdomen, pelvis and legs on day 1. This data will berelated to total activity of ¹¹¹ In administered and is a consistencycheck with the phantom data.

8. The geometric mean of the total cpm for whole body and each tissue ofinterest will be converted to ¹¹¹ In μCi/g. The conversion of cpm toactivity is given by the formula given below.

    A=SQRT(I.sub.a.I.sub.p)*1/[k*exp (-μT/2)]

where I_(a) and I_(p) are the cpm/pixel from anterior and posteriorcounts respectively, μT/2 is the attenuation half thickness for thedouble indium peak and will be derived from the water phantom studiesdescribed in step 6, and k is a camera sensitivity conversion factor ofthe cpm/pixel per μCi/g in air obtained from step 1.

9. For each set of images cpm/ROI will be converted to μCi/g in thattissue. Each set of data will then be decay corrected to yield thebiological uptake and clearance curve Y for each tissue.

10./The decay corrected data for each ROI will be plotted and fitted toan exponential clearance curve Y(t)=aexp(-λ_(b1) t) or an exponentialaccretion and clearance Y(t)=a(1-exp (-λ_(b2) t)·exp (-λ_(b1) t), whereY(t) is the tissue retention as a function of time, λ_(b1) and λ_(b2)are biological clearance and accretion constants, and a is a constantreflecting the peak fractional uptake in the tissue of interest.

11. The dose calculation for each tissue will be performed using theMIRD (Medical Internal Radiation Dose Committee) protocol. The radiationabsorbed dose D is given by

    D=(1/m)A(t)·Y(t)dt·ΣΔ.sub.i φ.sub.i

where A(t) is the physical decay curve for ⁹⁰ Y, Y(t), the retentioncharacteristics, m, the organ mass and ΣΔ_(i) φ_(i), the equilibriumdose constant for the radionuclide. For ⁹⁰ Y, ΣΔ_(i) φ_(i)=1.984(g)(cGy)/(μci)(hr). Since ⁹⁰ Y is a pure β⁻ -emitter, and theyield of Bremsstrahlung low, all the emitted radiation can be assumed tobe non-penetrating, i.e. deposited within the organ containing thatactivity. Extrapolation of the biological and physical clearance ratesfor normal organs will be assumed to parallel that of the blood.

12. Patients blood will be sampled 20 minutes after administration ofthe ¹¹¹ In and ⁹⁰ Y radiolabeled antibody and then daily thereafter.Pharmacokinetics and absorbed dose calculations for blood will bedetermined from direct measurements of the specific activity of ⁹⁰ Yfrom aliquots of the patients blood in a well scintillation counter.Comparison of the clearance curves for ¹¹¹ In and ⁹⁰ Y from the bloodwill be a good indicator of the relative stability of the two RICs.Appropriate detection procedures will be used to obtain independent ⁹⁰ Yand ¹¹¹ In information from the blood specimens. Alternative methods ofcalculating dosimetry estimates are within the scope of generalknowledge.

Radioimmunoscintigraphy and Radioimmunotherapy of Patients Using Anti-B1Antibody

In this study unlabeled B1 (2.5 mg/kg) is administered to patients 1hour before the administration of the radiolabeled B1 to minimize thenon-specific organ uptake of the radiolabeled B1. Antibody chelated with5 millicuries of ¹¹¹ In per dose or 20, 30, 40, or 50 mCi ⁹⁰ Y (or 5 mCiintervals as the dose nears the maximum tolerated dose) is administeredon B1-MX-DTPA (1 to 10 mg) mixed with unlabeled B1 for a total dose of10 mg-of antibody administered by intravenous infusion.

1. ¹¹¹ In-B1 and ⁹⁰ Y-B1 studies of Patient BLE

The urinary excretion of ¹¹¹ In was only 7.47% of total dose at 63 hourswith no unlabeled B1 carrier, but increased to 13.85% of total dose witha 1 mg/kg B1 dose. The estimated radiation dose from ¹¹¹ In was higherfor liver (1.22 cGy/mCi ¹¹¹ In or extrapolated 10.77 cGy/mCi ⁹⁰ Y) with2 mg ¹¹¹ In-B1-MX only than the estimated radiation dose for liver (0.62cGy/mCi ¹¹¹ In or extrapolated 5.86 cGy/mCi ⁹⁰ Y) when 1 mg/kg ofcarrier B1 was given (see TABLE 7). The dose estimates for the otherorgans were similar at both doses of B1. Carrier B1 (1 mg/kg) wasselected as the dosage for administration with the therapeutic dose of⁹⁰ Y B1-MX-DTPA.

Only a minor transient decrease in platelet concentration (nadir 113,000platelets/μL) was detected about 4 weeks after the administration of 20mCi ⁹⁰ Y-B1-MX-DTPA (see TABLE 8). There were no remarkable changes inother hematologic parameters for this patient after treatment (exceptthat WBC decreased slightly from the 4000 to 7000/uL range pre-treatmentto 3700 to 4800/uL range during the 4 weeks after treatment (TABLE 8).It was not necessary to re-infuse the patient's harvested autologousstem cells. The patient experienced light-headedness upon standingwithout loss of consciousness for a three day period, occurring about 2months after treatment.

The follow-up examinations of BLE one month after treatment with ⁹⁰Y-B1-MX-DTPA indicated a minor response in para-aortic and pelvic lymphnodes, but the right inferior gluteal node was estimated at 5.1×3.1×3.6cm by doppler flow. Follow-up at 2 months, 3 months, and 5 months hasindicated stable disease (right inferior gluteal node was estimated at4.4×3.2×4.4 cm by doppler flow after 5 months), but no further decreasein disease has been noted.

2. ¹¹¹ In-B1 and ⁹⁰ Y-B1 studies of Patient FPD

FPD entered on study after it was determined that he remained HAMAnegative despite prior exposure to murine monoclonal antibodies. Priorto imaging with ¹¹¹ In-B1-MX-DTPA left axillary node was 1.5 cm bypalpation with no abdominal abnormalities detectable by CT. Theparaaortic node at L4 was 1.0×0.8 cm by CT. The left Axillary nodeimaged by radioimmunoscintigraphy with ¹¹¹ In-B1-MX-DTPA at the nocarrier B1 dose and 1 mg/kg dose (83 mg B1). In addition a nodularpattern of uptake in the spleen that may be lymphoma was detected with¹¹¹ In-B1-MX-DTPA (first dose 2 mg ¹¹¹ In-B1-MX, second dose 1 mg/kg B1or 83 mg B1 plus 2 mg ¹¹¹ In-B1-MX).

The urinary excretion of ¹¹¹ In was 14.14% of total dose at 70 hourswith no unlabeled B1 carrier and 13.55% at 71.9 hours with a total dosewith a 1 mg/kg B1. The blood pool ¹¹¹ In was only 8.83% of the totaldose at 70.5 hours when no carrier B1 was given, but was 36.47% of thetotal dose when 1 mg/kg was given. The estimated radiation dose from ¹¹¹In was higher for spleen (0.08 cGy/mCi ¹¹¹ In or extrapolated 0.74cGy/mCi ⁹⁰ Y) with 2 mg ¹¹¹ In-B1-MX only than the estimated radiationdose for spleen (0.14 cGy/mCi ¹¹¹ In or extrapolated 1.36 cGy/mCi ⁹⁰ Y)when 1 mg/kg of carrier B1 was given (see TABLE 7). This increase in theradiation dose to total body is due to the increased fraction of ¹¹¹In-B1 in blood pool with time. The use of no carrier B1 lowered thebackground radiation to normal organs. FPD was given 20 mCi of ⁹⁰ Y-B1(2 mg B1-MX-DTPA without carrier B1.

No significant decrease in platelet concentration has been detected inthis patient post-treatment. There were no remarkable changes in otherhematologic parameters for this patient. It was not necessary tore-infuse the patient's harvested autologous stem cells. The patientexperienced no acute or chronic adverse reactions to the therapy.

The follow-up examinations of FPD one month and two months aftertreatment with ⁹⁰ Y-B1-MX-DTPA indicate partial responses at both times.After one month no palpable axillary nodes were found and thepara-aortic node at L4 was 0.5×0.4 cm by CT. After two months nopalpable axillary disease was detected and the paraaortic node was lessthan 0.3×0.3 cm by CT.

3. ¹¹¹ In-B1 and ⁹⁰ Y-B1 studies of Patient NWM

For patient NWM, prior to imaging with ¹¹¹ In-B1-MX-DTPA, left axillarynode was 1.5 to 2.0 cm by palpation with extensive abdominalabnormalities detectable by CT. Bilateral inguinal adenopathy wasdetected. Small (less than 1 cm) nodes in-the gastrohepatic ligament,iliac region, pancreatic, para-aortic chains were detected. Mediastinaland bilateral axillary nodes were also positive. Radioimmunoscintigraphywith ¹¹¹ In-B1-MX-DTPA at the no carrier B1 dose revealed localizationin the mediastinal, left axillary and mesenteric (parapancreatic) nodes.Imaging after ¹¹¹ In-B1-MX-DTPA plus the 1 mg/kg carrier dose of B1 (106mg) resulted in significant improvement in the number of nodes thatimaged. The carrier dose allowed images of the left supraclavicular,right and left Infraclavicular, mediastinal, left hilar, right and leftaxillary, paraaortic, mesenteric, right and left external iliac andright and left inguinal nodes. The urinary excretion of ¹¹¹ In was18.03% of total dose at 72 hours with no unlabeled B1 carrier and 36.08%at 71.2 hours with a total dose with a 1 mg/kg B1. The blood pool ¹¹¹ Inwas only 4.54% of the total dose at 72.5 hours when no carrier B1 wasgiven, but was 24.41% of the total dose when 1 mg/kg was given. Theestimated radiation dose from ¹¹¹ In was lower for total body (0.055Gy/mCi ¹¹¹ In or extrapolated 0.53 cGy/mCi ⁹⁰ Y) with 2 mg ¹¹¹ In-B1-MXonly than the estimated radiation dose for total body (0.092 cGy/mCi ¹¹¹In or extrapolated 0.88 cGy/mCi ⁹⁰ Y) when 1 mg/kg of carrier B1 wasgiven (see TABLE 7). This increase in the radiation dose to total bodyis due to the increased fraction of ¹¹¹ In-B1 in blood pool with time.The use of carrier B1 improved the targeting of lymph node sites ofdisease and increased the urinary clearance of ¹¹¹ In. NWM was given 20mCi of ⁹⁰ Y-B1 (2 mg B1-MX-DTPA) with 1 mg/kg (108 mg) of carrier B1.

4. ¹¹¹ In-B1 and ⁹⁰ Y-B1 studies of Patient JEF

For JEF, bone marrow involvement with tumor was estimated at 10% ofintra-trabecular space with 20% fat. Extensive abdominal nodalinvolvement including the retrocrural, right and left para-aortic,mesenteric, and right and left common, internal and external itiacnodes. The largest (left) para-aortic node measured 6.5×5 cm and theretrocrural and mesenteric nodes were <2 cm by CT. The spleen wasslightly enlarged. Imaging with ¹¹¹ In-B1-MX-DTPA (2.5 mg/kg B1antibody) resulted in an excellent correlation between theradioimmunoscintographs and the concomitant CT scan. The B1 antibodyscan agreed with the known sites of disease. Bone marrow also showedpositive on the scan.

JEF received 20.25 mCi of ⁹⁰ Y-B1-MX-DTPA with 2 mg/kg B1 monoclonalantibody (153 mg B1). JEF experienced no adverse reaction to either doseof B1 monoclonal antibody. The ⁹⁰ Y cleared from the blood at a rateconsistent with a blood half-life of 25.5 hours. The calculateddosimetry estimates estimated that the spleen received a radiation doseof 42.47 cGy/mCi ⁹⁰ Y-B1-MX-DTPA (TABLE 7). Liver and left kidney wereestimated to receive 22.47 cGy/mCi ⁹⁰ Y. The bone marrow was estimatedto receive 4.49 cGy/mCi ⁹⁰ Y based upon a single point bone biopsy.

Autologous bone marrow was re-infused into JEF 18 days after the dose of⁹⁰ Y-B1-MX-DTPA. Twenty-four days after treatment with ⁹⁰ Y-B1-MX-DTPAthe patient was judged to have had a minor response with some decreasein splenomeglia and the left para-aortic node had decreased from 6.5×5cm to 5.5×4.5 cm measured by CT. Restaging of the patient 57 days aftertreatment indicated-a further minor response with the L paraaortic nodedecreasing to 4×5 cm. Re-staging at 86 days indicated no furtherreduction in disease, but stable disease (left para-aortic node 3.5×4.8cm). This patient had progressive disease prior to treatment with ⁹⁰Y-B1.

JEF experienced a grade III decrease in platelet counts of approximately31 days duration. The observed nadir of 26,000 platelets/μL was at day24 after treatment. JEF continued with platelet counts in the 31,000 to54,000/μL at least through day 117 after treatment. JEF experienced nosignificant medical consequences to this lowered platelet levels andreturned to her employment at approximately day 60 after treatment.

5. ¹¹¹ In-B1 and ⁹⁰ Y-B1 studies of Patient BAH

Bone marrow involvement with tumor was estimated at 10% ofintra-trabecular space with 20% fat. Extensive abdominal nodalinvolvement including the retrocrural, right and left para-aortic,mesenteric, and right and left common, internal and external iliacnodes. The largest (left) para-aortic node measured 6.5×5 cm and theretrocrural and mesentenic nodes were <2 cm by CT. The spleen wasslightly enlarged. Imaging with ¹¹¹ In-B1-MX-DTPA (2.5 mg/kg B1antibody) resulted in good tumor localization especially in the axillarynode. BAH received 20.8 mCi of ⁹⁰ Y-B1-MX-DTPA with 2 mg/kg B1monoclonal antibody 1150 mg B1). BAH experience no adverse reaction toeither dose of B1 monoclonal antibody. The ⁹⁰ Y cleared from the bloodat a rate consistent with a blood half-life of 27.5 hours. Thecalculated dosimetry estimates estimated that the spleen received aradiation dose of 13.63 cGy/mCi ⁹⁰ Y-B1-MX-DTPA. Liver exposure wasestimated at 16.42 cGy/mCi ⁹⁰ Y. The right kidney was estimated at 23.25cGy/mCi and left kidney at 21.04 cGy/mCi ⁹⁰ Y. The bone marrow wasestimated to receive 16.27 cGy/mCi ⁹⁰ Y based upon a single point bonebiopsy.

Autologous bone marrow was re-infused into BAH 17 days after the dose of⁹⁰ Y-B1-MX-DTPA. Twenty-three days after treatment with ⁹⁰ Y-B1-MX-DTPAthe patient was judged to have progression of disease (especiallyaxillary node which increased from 4×4 cm to 4.8×4.8×7 cm by CT. Thehydronephrosis was stable with a slight increase in the liver lesions.Continued progression was also noted in the axillary node at 49 days(7.5×7.5 cm by CT, 10×8 cm by physical examination) with extensivepleural effusion with acute hydronephrosis and chronic hydronephrosis onthe left kidney. BAH was lost to study and further interpretation. BAHdeveloped a candida genitourinary infection secondary to thehydronephrosis. The initial interpretation was that the hydronephrosiswas attributable to bulky blockage of the ureters. The patient was given2000 cGy of external beam therapy to the abdomen, pelvis and axilla. Thehydronephrosis was subsequently attributed to blockage by stones and ordebris and was successfully treated.

BAH experienced a grade IV decrease in platelets with a nadir of 13,000platelets/μL and a grade III decrease in white blood cells (1000 WBC/μL)39 days after therapy with ⁹⁰ Y-B1. Because of the external beamradiation therapy it was not possible to further assess thehematological toxicity of the ⁹⁰ Y-B1 dose. The patient did requireperiodic platelet support for an extended period.

Conclusions:

Significant toxicities have not been observed at the 13 and 20 mCi levelof ⁹⁰ Y-B1-MX-DTPA. Four patients treated to date have shown 2 minorresponses, one partial remission and one complete response.

Two additional patients have completed study. One additional patientthat completed study had a partial response, but experienced grade IIIsuppression of platelets. The other additional patient had a partialremission, radiotherapy was given to the brain of this patient after newdisease appeared and the patient is now in a complete remission status.A third additional patient, who is thus patient number 7, received ⁹⁰Y-B1-MX-DTPA therapy, but could not be evaluated for toxicity due toexternal beam radiation therapy that was given to treat hydronephrosissymptoms. This seventh patient's disease continued to progress, exceptin the field of the external beam therapy. Patient number 7 failed todemonstrate adequate tumor localization with ¹¹¹ In-B1-MX and thus didnot enter the therapy phase of the study.

                                      TBLE 7                                      __________________________________________________________________________    Dosees, radiochemical purities, imaging and toxicity for 6 patients           on .sup.90 Y-B1. Patients were imaged with .sup.111 Indium-B1-MX-DTPA. A      dose of                                                                       B1 (2.5 mg/kg body weight at DFCI or 0 or 1 mg/kg body weight) B1             was administered just prior to administration of the imaging or               therapeutic doses. Disease sites detected by                                  radioimmunoscintigraphy/number of known sites of disease by all               other methods. No adverse reactions were observed upon                        administration of B1/.sup.111 In-B1-MX-DTPA drug.                                        1-001                                                                              1-002 1-003  1-004 2-001                                                                              2-002 2-003  2-004                    Patient number                                                                           JEF  BAH   SJM    NJP   BLE  FPD   NWM    SR                       __________________________________________________________________________    90-Yttrium-B1 (mCi)/                                                                     20.3 mCi                                                                           21.2 mCi                                                                            No 90-Y-B1   13.6 mCi                                                                           13.5 mCi                                                                            13.7 mCi                                                                             21.6                     mg cold B1 153 mg                                                                             150 mg                                                                              dose         85 mg      106 mg mCi                      RIS detected                       1/10 2/2   2/9    ?                        sites/known sites (no                                                         cold B1)                                                                      RIS detected          4/4    0/1   1/10 2/2   9/9    ?                        sites/known sites     1                       5                               (with cold B1)        unconfirmed             unconfirmed                                           site in lung            sites**                         Clinical Response                                                                        Stabile                                                                            Progression                                                                         No therapy                                                                           Partial                                                                             Minor                                                                              Complete                                                                            Partial                                                                              Minor                               Disease           Remission                                                                           Response                                                                           Remission                                                                           Remission@                                                                           response                 __________________________________________________________________________     @ Patient subsequently received radiotherapy to brain after new brain met     appeared, Patient now in complete remission                                   *Patient had contiguous lymphomatous involvement of the retroperitoneal       and mesenteric lymphadenopathy, there was an excellent correlation with       concomitant CT scan showing radiolabeled monoclonal antibody in sites of      anatomically defined lymphomatous involvement. Onse site in knee remained     unexplained.                                                                  **5 unconfirmed lymph node sites were detected by RIS.                        ***Chills and slight lightheadedness reported during only 1 of 3              administrations of B1.                                                   

                                      TABLE 8                                     __________________________________________________________________________    Dosimetry calculations for organs total body and urinary clearance            estimates from .sup.90 Y-B1 study based upon                                  extrapolation of .sup.111 Indium-B1-MX-DTPA distribution and                  pharmacokinetics and clearance. Radiation dose is                             cGy/mCi .sup.90 Yttrium.                                                                1-001 1-002 2-001 2-001  2-002 2-002  2-003  2-003                            JEF   BAH   BLE   BLE    FPD   FPD    NWM    NWM                    Patient number                                                                          2.5 mg/kg                                                                           2.5 mg/kg                                                                           0 mg B1                                                                             1 mg/kg                                                                              0 mg B1                                                                             1 mg/kg                                                                              0 mg B1                                                                              1                      __________________________________________________________________________                                                           mg/kg                  Total Body                                                                              2.32  2.08  1.44  1.51   0.74  1.36   0.53   0.88                   Liver     22.47 16.42 10.77 5.85   7.06  6.43   7.68   5.61                   Spleen    42.47 13.63 4.84  4.27   17.23 7.41   10.76  11.28                  Heart     12.79 18.54 11.82 12.75  6.14  8.41   1.73   5.47                   Right Kidney                                                                            14.47 23.25                                                         Left Kidney                                                                             22.47 21.04                                                         Lumbar Spine          2.94  3.62                                              Lung      14.32 8.63                                                          Bone Marrow                                                                             4.49  16.27                                                         (biopsy)                                                                      Blood     6.96  8.07                                                          Blood Biol T1/2 90-                                                                     25.5  27.5                                                          Y (hrs)                                                                       Urinary excretion     7.5% TD at                                                                          13.9% TD                                                                             14.1% TD                                                                            13.6% TD                                                                             18.0% TD                                                                             36.1% TD at            111-In (% TD)         63 hr at 63 hr                                                                             at 70 hr                                                                            at 72 hr                                                                             72 hr  72 hr                  Urinary excretion           12.2% TD                                                                             9.2% TD at          16.1% TD at            90-Y (% TD)                 at 87 hr                                                                             69 hr               71                     __________________________________________________________________________                                                           hr                 

EXAMPLE IV SENSITIZATION OF LYMPHOMA CELLS BY ANTI-CD20 ANTIBODY

Apoptosis is a phenomenon in cell biology, wherein a cell becomescommitted to its own destruction. A cell which is apoptoticdisplays-characteristic changes in metabolism, which ultimately resultin fragmentation of cellular DNA and lysis of the cell.

The excellent results described above for radioimmunotherapy using ananti-CD20 antibody might be due, in part, to synergism in the inductionof apoptosis by binding of the anti-CD20 antibody and the irradiation ofthe tumor cell. The evidence for this hypothesis comes primarily fromthe tumor responses observed to the trace-labeled antibody administeredfor imaging purposes.

Given such synergism in the induction of apoptosis by binding ofunlabeled anti-CD20 and some second insult to the cell, it is expectedthat administration of an anti-CD20 antibody could be combined with avariety of secondary treatments to achieve the same synergism. Forexample, binding of a second antibody, directed against a differentantigen than CD20, that is conjugated to a radionuclide would providethe same synergistic second insult to the tumor cell as is provided byan anti-CD20 radioimmunoconjugate.

Also, a similar effect could be provided by external beam irradiation.If external beam irradiation is used, then the dose to be administeredshould be in the range of 100 to 250 cGy to whole body if bone marrowreplacement support is not contemplated. However, if bone marrowreplacement is used as an adjunct therapy, doses as high as 1000 cGy towhole body could be used. Such a large dose would likely be administeredin a series of fractional doses.

Finally, one can consider inducing the apoptosis events synergisticallyby administering a chemotherapeutic agent. In choosing thechemotherapeutic agent, one would preferably employ a drug which is aDNA alkylating agent, such as cyclophosphamide or chlorambucil. Anotherpreferred class of drugs are the antimetabolites, such as methotrexate.In particular, cyclophosphamide, chlorambucil, doxorubicin andmethotrexate are preferred to be administered for this mode of therapy.

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What is claimed is:
 1. A method for immunotherapy of B-cell lymphoma,which comprises:(i) administering to a patient an imaging effectiveamount of an antibody, or a Fab, Fab' or F(ab')₂ portion thereof, tracelabelled with a first radiolabel, which binds to CD20 antigen present onthe surface of cells of said B-cell lymphoma; (ii) imaging thedistribution of said labelled antibody, or a Fab, Fab' or F(ab')₂portion thereof of step (i), within the body of the patient; (iii)administering to the patient an amount of the antibody or a Fab, Fab' orF(ab')₂ portion thereof of step (i) in unlabelled form, which binds toCD20 antigen present on the surface of cells of said B-cell lymphoma,said amount effective for blocking non-tumor binding sites for anantibody, or Fab, Fab' or F(ab')₂ portion thereof effective for treatingB-cell lymphoma, within the body of the patient; and (iv) administeringto the patient a radioimmunotherapeutically effective amount fortreating B-cell lymphoma of said antibody, or a Fab, Fab' or F(ab')₂portion thereof of step (i), this is labelled with said first radiolabelor with a different radiolabel wherein the amount of radioactivity isless than the amount that provides irradiation at a dose which causesmyelosuppression severe enough to require the reintroduction ofhematopoietic stem cell into the patient in order for the to recoverhematopoietic function after administration of said antibody or Fab,Fab' or F(ab')₂ portion thereof which is administered in saidradioimmunotherapeutically effective amount.
 2. The method of claim 1,wherein said antibody is labelled with a β⁻ emitter.
 3. The method ofclaim 2, wherein said antibody is labelled with an isotope selected fromthe group consisting of ¹³¹ I, ⁹⁰ Y and ¹⁸⁶ Re.
 4. The method of claim1, wherein the amount of radioactivity for therapy provides irradiationat a dose in the range of 10 to 200 cGy to the whole body of saidpatient per administration.
 5. The method of claim 4, wherein the amountof antibody administered in step (i) is the same as the amountadministered in steps (iii) and (iv).
 6. The method of claim 4, whereinthe antibody, or Fab, Fab' or F(ab')₂ fragment thereof of step (i), andthe antibody, Fab, Fab' or F(ab')₂ fragment thereof of step (iv), arelabeled with ¹³¹ I.
 7. The method of claim 1, wherein the amount ofradioactivity for therapy provides irradiation at a dose in the range of25 to 150 cGy to the whole body of said patient per administration. 8.The method of claim 1, wherein the antibody administered in step (i) islabelled with ⁹⁹ Tc or ¹¹¹ In and wherein the antibody administered instep (iv) is labelled with an isotope selected from the group consistingof ¹³¹ I, ⁹⁰ Y and ¹⁸⁶ Re.
 9. The method of claim 8, wherein the amountof antibody administered in step (i) is the same as the amountadministered in steps (iii) and (iv).
 10. The method of claim 1, whereinan amount of radioactivity between 5 and 250 mCi is administered to thepatient in step (iv).
 11. The method of claim 1, wherein the amount ofantibody administered in step (i) is the same as the amount administeredin steps (iii) and (iv).
 12. The method of claim 11, wherein theantibody, or Fab, Fab' or F(ab')₂ fragment thereof in step (i), and theantibody, Fab, Fab' or F(ab')₂ fragment thereof in steps (iii) and (iv),are labeled with ¹³¹ I.
 13. The method of claim 1, wherein the antibody,or Fab, Fab' or F(ab')₂ fragment thereof of step (i), and the antibody,Fab, Fab' or F(ab')₂ fragment thereof of step (iv) are labeled with ¹³¹I.
 14. A method for immunotherapy of a neoplasm of B cell lineage, whichcomprises:(i) administering to a patient an imaging effective amount ofan antibody, or a Fab, Fab' or F(ab)₂, portion thereof, which binds toCD20 antigen present on the surface of cells of B lineage that istrace-labelled with a first radiolabel; (ii) imaging the distribution ofsaid labelled antibody, or Fab, Fab' or F(ab')₂ portion thereof of step(i), within the body of the patient; (iii) administering to the patientan amount of the antibody, or a Fab, Fab' or F(ab')₂ portion thereof ofstep (i) in unlabelled form, said amount being effective for blockingnon-specific binding sites for an antibody effective for treating saidneoplasm of B-cell lineage within the body of the patient; and (iv)administering to the patient a radioimmunotherapeutically effectiveamount for treating said neoplasm of B-cell lineage of said antibody, ora Fab, Fab' or F(ab')₂ portion thereof of step (i), which binds to CD20antigen present on the surface of said cells of B lineage, that islabelled with said first radiolabel or with a different radiolabelwherein the amount of radioactivity is less than the amount thatprovides irradiation at a dose which causes myelosuppression severeenough to require the reintroduction of hematopoietic stem cells intothe patient in order for the patient to recover hematopoietic functionafter administration of said antibody, or Fab, Fab' or F(ab')₂ portionthereof which is administered in said radioimmunotherapeuticallyeffective amount.
 15. The method of claim 14, wherein the amount ofantibody administered in step (i) is the same as the amount administeredin each of steps (iii) and (iv).
 16. A method for immunotherapy ofB-cell lymphoma, which comprises:(i) administering to a patient animaging effective amount of an antibody, or a Fab, Fab' or F(ab')₂portion thereof, which binds to CD20 antigen present on the surface ofcells of said B-cell lymphoma that is trace labelled with a radiolabel;(ii) imaging the distribution of said labelled antibody, or Fab, Fab' orF(ab')₂ portion thereof of step (i), within the body of the patient;(iii) administering to the patient an amount of the antibody, or a Fab,Fab' or F(ab')₂ portion thereof of step (i) in unlabelled form, whichbinds to CD20 antigen present on the surface of cells of said B-celllymphoma, said amount being effective for blocking non-tumor bindingsites for an antibody effective for treating B-cell lymphoma within thebody of the patient; and (iv) administering to the patient aradioimmunotherapeutically effective amount for treating B-cell lymphomaof said labelled antibody, or a Fab, Fab' or F(ab)₂ portion thereof ofstep (i), which binds to CD20 antigen present on the surface of cells ofsaid B-cell lymphoma, wherein the amount of radioactivity is less thanthe amount that provides irradiation at a dose which causesmyelosuppression severe enough to require the reintroduction ofhematopoietic stem cells into the patient in order for the patient torecover hematopoietic function after administration of theradioimmunotherapeutically effective amount of said antibody, or Fab,Fab' or F(ab')₂ portion thereof which is administered in saidradioimmunotherapeutically effective amount.
 17. The method of claim 16,wherein the amount of antibody administered in step (i) is the same asthe amount administered in each of steps (iii) and (iv).
 18. A methodfor immunotherapy of B-cell lymphoma, which comprises:(i) administeringto a patient an effective amount of unlabeled antibody, or a Fab, Fab'or F(ab')₂ portion thereof, which binds to CD20 antigen present on thesurface of cells of said B-cell lymphoma, said amount effective forblocking non-tumor binding sites for said antibody in the body of saidpatient; (ii) administering an imaging effective amount of the antibody,or a Fab, Fab' or F(ab')₂ portion thereof of step (i), which binds toCD20 antigen present on the surface of cells of said B-cell lymphoma,that is trace-labelled with a first radiolabel; (iii) imaging thedistribution of said labelled antibody, or a Fab, Fab' or F(ab')₂portion thereof of step (ii), within the body of the patient; (iv)administering to the patient an amount of the unlabelled antibody, or aFab, Fab' or F(ab')₂ portion thereof, which binds to CD20 antigenpresent on the surface of cells of said B-cell lymphoma, said amounteffective for blocking non-tumor binding sites for an antibody effectivefor treating B-cell lymphoma, or Fab, Fab' or F(ab')₂ portion thereof,within the body of the patient; and (v) administering to the patient aradioimmunotherapeutically effective amount for treating B-cell lymphomaof said antibody, or a Fab, Fab' or F(ab')₂ portion thereof, of step(ii) which binds to CD20 antigen present on the surface of cells of saidB-cell lymphoma, that is labelled with said first radiolabel or with adifferent radiolabel wherein the amount of radioactivity is less thanthe amount that provides irradiation at a dose which causesmyelosuppression severe enough to require the reintroduction ofhematopoietic stem cells into the patient in order for the patient torecover hematopoietic function after administration of said antibody orFab, Fab' or F(ab')₂ portion thereof which is administered in saidradioimmunotherapeutically effective amount.
 19. The method of claim 18,wherein the amount of antibody administered in step (ii) is the same asthe amount administered in step (iv).
 20. The method of claim 19,wherein the antibody, or Fab, Fab' or F(ab')₂ fragment thereof of step(ii), and the antibody, Fab, Fab' or F(ab')₂ fragment thereof of step(iv), are labeled with ¹³¹ I.
 21. The method of claim 18, wherein theantibody, or Fab, Fab' or F(ab')₂ fragment thereof of step (ii), and theantibody, Fab, Fab' or F(ab')₂ fragment thereof of step (v), are labeledwith ¹³¹ I.
 22. A method for immunotherapy of B-cell lymphoma, whichcomprises:(i) administering to a patient a first amount of an unlabeledantibody, or a Fab, Fab' or F(ab')₂ portion thereof, which binds to CD20antigen present on the surface of cells of said B-cell lymphoma, saidfirst amount effective for blocking non-tumor binding sites for saidantibody in the body of said patient; (ii) administering to a patient animaging effective amount of said antibody, or a Fab, Fab' or F(ab')₂portion thereof of step (i), which is trace-labeled with a firstradiolabel; (iii) imaging the distribution of said labelled antibody, orFab, Fab' or F(ab')₂ portion thereof of step (ii), within the body ofthe patient; (iv) administering to the patient a second amount of theunlabelled antibody, or a Fab, Fab' or F(ab')₂ portion thereof, as wasused in step (i), said second amount effective for blocking non-tumorantibody binding sites within the body of the patient; and (v)administering to the patient a radioimmunotherapeutically effectiveamount for treating B-cell lymphoma of said antibody, or a Fab, Fab' orF(ab)₂ portion thereof of step (ii), which binds to CD20 antigen presenton the surface of cells of said B-cell lymphoma, that is labelled withsaid first radiolabel or with a different radiolabel wherein the amountof radioactivity is less than the amount that provides irradiation at adose which causes myelosuppression severe enough to require thereintroduction of hematopoietic stem cells into the patient in order forthe patient to recover hematopoietic function after administration ofsaid antibody, or Fab, Fab' or F(ab')₂ portion thereof which isadministered in said radioimmunotherapeutically effective amount. 23.The method of claim 22, wherein the amount of antibody administered instep (ii) is the same as the amount administered in the therapeutic step(iv).
 24. The method of claim 23, wherein the antibody, or Fab, Fab' orF(ab')₂ fragment thereof of step (ii), and the antibody, Fab, Fab' orF(ab')₂ fragment thereof of step (iv), are labeled with ¹³¹ I.
 25. Themethod of claim 22, wherein the antibody, or Fab, Fab' or F(ab')₂fragment thereof of step (ii), and the antibody, Fab, Fab' or F(ab')₂fragment thereof of step (v), are labeled with ¹³¹ I.